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TH ms ,5"?an Y
5 MlChlgan State

205;? UnIVersity

ﬂ

This is to certify that the
dissertation entitled

YOGURT FORTIFICATION WITH
PREDIGESTEDIGERMINATED WHOLE SOYBEAN
POWDER FOR ENHANCED THERAPEUTIC BENEFITS

presented by

OBIANUJU NWAMAKA NSOFOR

has been accepted towards fulﬁllment
of the requirements for the

DOCTORAL degree in FO,QD SCIENCE

 

 

yﬁWﬂ/IIIH /

a' r rofesEor’Wure
393‘ \qu 042

Date

MSU is an Afﬁrmative Action/Equal Opportunity Employer

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 K:IProlecc&Pres/CIRC/DateDue.indd

YOGURT F ORTIFICATION WITH PREDIGESTEDIGERMINATED
WHOLE SOYBEAN POWDER FOR ENHANCED
THERAPEUTIC BENEFITS

By

Obianuju Nwamaka Nsofor

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Food Science

2008

ABSTRACT

YOGURT FORTICATION WITH PREDIGESTED/GERMINATED WHOLE
SOYBEAN POWDER FOR ENHANCED THERAPEUTIC BENEFITS

By

Obianuju Nwamaka Nsofor

Growth (CFU/ml), changes in pH and titratable acidity (%TA) measurements were
used to study culture activities and grth of 3 lactic acid bacteria (LAB) namely
Streptococcus salivarius subsp. thermophilus (St 133), Lactobacz'llus delbruekii subsp.
bulgaricus (Lr 78) and Lactobacillus acidophilus (La NCFM). These cultures were
grown in blends (3 :7, 1:1, 7:3) of reconstituted non-fat dry milk (N FDM) and germinated
(GSP) or non-germinated (N GSP) whole soy powder obtained from three soybean
varieties (Vinton 81, DF 222 and E05276-T). Cultures grown in most milk blends that
contained both germinated and non-germinated whole soy powder gave low pH, high
%TA and growth values. The lowest pH and highest %TA and growth were obtained
when the cultures were grown in 1:1 NFDM+GSP or NGSP blend. Cultures grown in
germinated whole soy powder blends produced more acid and growth (pg .001) than its
non-germinated counterparts. The 1:1 blends of NFDM and GSP or NGSP were utilized
to make low-fat Swiss-style strawberry yogurt. Sensory evaluation by 112 untrained
panelists showed that there was no consumer preference for cow’s milk yogurt over
whole soy- fortiﬁed cow’s milk yogurt.

The concentrations of isoﬂavone isomers genistein, daidzein, genistin and daidzin

were determined in raw soy powder (RSP), NGSP and GSP of Vinton 81 , DF 222 and

E05276-T soybean varieties using reverse-phase high-performance liquid
chromatography (HPLC). Stachyose (oligosaccharide) contents of NGSP and GSP were
analyzed using HPLC. The combined four isoﬂavone isomers and stachyose
concentrations were highest in non-germinated (soaked) DF 222. All the germinated and
non-germinated soy powders contained high quantities of isoﬂavone irrespective of
soybean variety. Germinated soy powders had signiﬁcantly lower amount of stachyose
than non-germinated powders (p < 0.001). Also HPLC analysis of yogurt samples
showed that total isoﬂavone contents increased from 1St to 6th week of cold storage. The
genistein and daidzein contents of the soy-fortiﬁed yogurts remained the same throughout
the 6 weeks of storage.

Shelf life studies of the yogurt samples indicated that growth and viability of all _
the cultures were above 107 CFU/ g of yogurt. A concentration of at least 106 viable
probiotic cultures is needed in fermented foods in order to exert health beneﬁts to the
consumers. Soy-fortiﬁed yogurts had the highest concentration of cell counts, which were

statistically different from all dairy or soy yogurts.

To my late parents
Herbert and Mercy Ikpeze who taught me the value of education and sacriﬁced so much
to make sure all their 10 children were educated. Thanks mom for teaching me patience
and the importance of always running life’s race in my assigned lane so that I will never
be disqualiﬁed.
And
To my late mother-in-law
Florence Anamaleze—Nsofor who showered me with so much love that surpasses a

mother’s love for her child.

iv

ACKNOWLEDGEMENTS

This dissertation would not have been possible without the assistance and support
ﬁ'om many individuals. I would like to acknowledge and wholeheartedly thank Michigan
Soybean Promotion Committee for funding this work. I am grateful to the chairperson,
Department of Food Science and Human Nutrition, Dr G. Strasburg for his assistance and
interest throughout the course of my study here. To Dr Zey Ustunol, my major professor
(adviser), mentor and ﬁ'iend. Words cannot express my gratitude to you for all your
constructive criticisms and encouragement especially when I was told by one of your
peers that my PhD quest was ﬁrtile after staying out of school for so many years. You
provided enthusiasm, continued commitment and a realistic perspective that were crucial
during my study. Thanks to my committee members Drs J. Partridge, M. Bennink and D.
Wang for their time and assistance.

To my family, I owe the most. My children Leslie Jr, Valentine and Stephanie, and
my niece Diane, lights of my life, always able to lift my spirit so simple. You ﬁll me with
a sense of pride that far exceeds any accomplishment in life. My husband Leslie, you are
my best friend and partner forever in every sense of the word. You not only inspire me to
be a better person, you still love me as much even when I fall short of my goals. I have a
better time with you than anyone else in the world and this is true even when we disagree.
Thank you for all your on-going and continual support. Whatever life may bring on our
journey together, you are my biggest cheerleader. My siblings Uche, Ngozi, Obika,
Nnenne, Chinwude (late), Nkem, Ifeoma, Okey and Arinze,you guys always told me to

follow my dreams and you helped make them come true. You never stopped spoiling me

as your kid sister. Thank you Mma Nwokocha for your prayers and encouragement and
to Chinyere Tobias for your enduring support throughout my studies.

I owe a debt of gratitude to Drs Pestka and Dolan for giving me unlimited access
to their labs and equipment. To Dr Janice Harte for her encouragement and advice on
sensory work and Dave Main (Dept. of Animal Science, MSU) for his expertise on
nutrient analysis of soy powders and yogurt samples. Many thanks go to Rodney Clark
and John Engstrom for their help during yogurt manufacturing. Thanks to my colleagues
and friends especially Dozie Amuzie, Sara Magers, Katie Barret, Lori Madaj, Laura
Vines, Becky Kyong, Aileen Tanojo, Kerri Harris, Lindsey Keskinen and everybody that
contributed to my success here. Finally to my maker and Almighty God, no step was

taken without your knowledge. Dreams can, and do come true.

“Learning is the fountain of youth. No matter how old you are, you musn’t stop

growing.”

365 Tao: Daily Meditations

Deng Ming-Dao

Harper, San Francisco, 1992.

vi

TABLE OF CONTENTS

LIST OF TABLES .............................................................................. xi
LIST OF FIGURES ............................................................................. xiv
INTRODUCTION .............................................................................. 1
CHAPTER 1
LITERATURE REVIEW ...................................................................... 4
1.1 COW’S MILK YOGURT ........................................................ 4
1.1.1 Composition and nutritional value of milk ........................ 4
1.1.2 Nutritional and health beneﬁts of yogurt ........................... 5
1.1.2.1 Deﬁnition and history of yogurt ............................ 5
1.1.2.2 Special ingredients of yogurt ................................ 6
1.1.2.3 Biodefense properties of yogurt ............................. 8
1.2 SOYBEAN PRODUCT ......................................................... 1 1
1.2.1 Composition and nutritional value of soybean and soy
products ................................................................. 11
1.2.1.1 Chemical composition of soybean ......................... 12
1.2.1.2 Soybean protein .............................................. 13
1.2.1.3 Soy isoﬂavones .............................................. 15
1.2.1.4 Soy oligosaccharides ........................................ 19
1.2.1.5 Soybean germination ........................................ 21
1.2.1.6 Soy and health ................................................ 24
1.2.1.7 Soy products ................................................... 26
1.3 PROBIOTICS AND HEALTH ................................................ 26
CHAPTER 2

GROWTH AND ACTIVITY OF LACTIC ACID BACTERIA (LAB) AND A
PROBIOTIC IN RECONSTITUTED GERMINATED WHOLE SOY POWDER (GSP),

NON-GERMINATED WHOLE SOY POWDER (NGSP) AND NON-FAT DRYMILK
(NFDM) + GSP OR NGSP

2.1 ABSTRACT ........................................................................ 33
2.2 INTRODUCTION ................................................................. 34
2.3 MATERIALS AND METHODS ................................................ 36
2.3.1 Materials .................................................................. 36
2.3.1.1 Cultures ........................................................... 36
2.3.1.2 Soybean ........................................................... 37
2.3.2 Germinated and non-germinated whole soy powder
preparation ............................................................... 37
2.3.3 Growth and activity evaluation ........................................ 46
2.3.4 Statistical analysis ....................................................... 47
2.4 RESULTS AND DISCUSSION ................................................. 48

vii

CHAPTER 3
DEVELOPMENT AND PROPERTIES OF YOGURT FROM BLENDS OF COW’S
MILK AND WHOLE SOYMILK BASE FOR CONSUMER ACCEPTANCE

3.1 ABSTRACT ........................................................................... 76
3.2 INTRODUCTION ................................................................... 77
3.3 MATERIALS AND METHODS .................................................. 79
3.1.1 Low fat yogurt formulation and manufacture ......................... 79
3.1.2 Screening of Swiss-style strawberry ﬂavored low-fat yogurt
by experienced consumers .............................................. 82
3.1.3 Sensory evaluation of Swiss-style strawberry ﬂavored low-fat
yogurt ....................................................................... 82
3.1.4 Proximate analysis of yogurt samples .................................. 83
3.1.5 Statistical analysis ........................................................ 84
3.4 RESULTS AND DISCUSSION ................................................... 84
3.4.1 Sensory evaluation of yogurt samples .................................. 84
3.4.2 Effect of whole soy powder fortiﬁcation on pH of yogurts .......... 87
3.4.3 Nutrient composition of yogurt samples ................................ 88
3.4.3.1 Effect of soy fortiﬁcation on protein content of yogurt. .....88
3.4.3.2 Effect of soy fortiﬁcation on fat content of yogurt ........... 90
3.4.3.3 Effect of soy fortiﬁcation on carbohydrate content of
yogurt ............................................................... 90
3.4.3.4 Effect of soy fortiﬁcation on the ash and dietary ﬁber
contents of yogurt ................................................ 91
CHAPTER 4

PRODUCTION AND CONCENTRATION OF GENISTEIN, DAIDZEIN, GENISTIN,
DAIDZIN AND STACHYOSE IN PREDIGESTED/GERMINATED AND NON-
GERMINATED SOY POWDER

4.1 ABSTRACT ...................................................................... 93
4.2 INTRODUCTION .............................................................. 94
4.3 MATERIALS AND METHODS ............................................. 97
4.3.1 Materials ................................................................. 97
4.3.1.1 Chemicals and solutions .................................... 97
4.3.1.2 Instrumentation ............................................... 98
4.3.2 Methods ................................................................... 98
4.3.2.1 Calibration curves and calculation of standard
solutions ......................................................... 98
4.3.2.2.Proximate analysis of soy powders ......................... 102
4.3.2.3 Isoﬂavone extraction .......................................... 102
4.3.2.4 Reverse-phase high performance liquid
chromatography (HPLC) of isoﬂavones .................... 103
4.3.2.5 Reverse-phase high performance liquid chromatography
(HPLC) of stachyose .......................................... 103
4.3.3 Statistical analysis ...................................................... 105

viii

4.4 RESULTS AND DISCUSSION ............................................. 105
4.4.1 Effect of germination on compositional analysis of soybean

powder ..................................................................... 105
4.4.2 Effect of germination on total isoﬂavone content .................... 108
4.4.3 Effects of germination on Genistein and Genistin contents... ......109
4.4.4 Effects of germination on Daidzein and Daidzin contents ......... 111
4.4.5 Effects of germination on Stachyose contents ........................ 118

CHAPTER 5

EFFECT OF PROCESSING AND REFRIGERATED STORAGE ON ISOFLAVONE
AND STACHYOSE CONTENTS OF YOGURT FORTIFIED WITH NON-
GERMINATED AND GERMINATED/PREDIGESTED WHOLE SOY POWDER

5.1 ABSTRACT ........................................................................... 120
5.2 INTRODUCTION ................................................................... 121
5.3 MATERIALS AND METHODS .................................................. 123
5.3.1 Yogurt samples ........................................................... 123

5.3.2 Instrumentation and solutions .......................................... 124

5.3.3 Extraction and Evaporation ............................................ 125

5.3.4 HPLC Analysis .......................................................... 125

5.3.5 Statistical Analysis ...................................................... 126

5.4 RESULTS AND DISCUSSION .................................................. 126

5.4.1 Effects of soy fortiﬁcation on total isoﬂavone content of yogurt. . . 126
5.4.2 Effects of soy fortiﬁcation on Genistein and Genistin content of

yogurt ........................................................................ 135
5.4.3 Effects of soy fortiﬁcation on Daidzein and Daidzin contents of
yogurt ........................................................................ 138
5.4.3 Effects of soy fortiﬁcation on Stachyose content of yogurt ....... 143
CHAPTER 6
SHELF LIFE STUDIES AND VIABILITY OF WHOLE SOY-FORTIFIED YOGURTS
STORED AT 4 °C
6.] ABSTRACT ............................................................................ 147
6.2 INTRODUCTION ..................................................................... 148
6.3 MATERIALS AND METHODS .................................................... 150
6.3.] Media Preparation ......................................................... 150
6.3.2 Enumeration of Lactic Acid Bacteria ................................. 151
6.3.3 Statistical analysis ....................................................... 152
6.4 RESULTS AND DISCUSSION, ...................................................... 152
6.4.1 Viability of microorganisms in yogurts during cold storage ...... 152
6.4.1.1 Viability of Lactobacillus delbreuckii subsp.bulgaricus
during cold storage ............................................. 153
6.4.1.2 Viability of Streptococcus thermophilus during cold
storage ........................................................... 157
6.4.1.3 Viability of Lactobacillus acidophilus during cold
storage ............................................................ 160
6.4.2 pH changes of yogurt samples during cold storage ................ 164

ix

CONCLUSIONS ................................................................................. 1 68

APPENDICES .................................................................................... 1 71
APPENDIX 1 Questionnaire for experienced yogurt screeners ....................... 172
APPENDIX 2 Advertisement ................................................................. 173
APPENDIX 3 UCHRIS Approval ........................................................... 174
APPENDIX 4 Consent form .................................................................. 175
APPENDIX 5 Questionnaire for untrained panelists .................................... 177
REFERENCES .................................................................................... 180

LIST OF TABLES

Table 1.1 Effects of soybean varieties and growing environments on soybean
protein, the protein and total solid contents soymilk .............................. 14

Table 1.2 Total isoﬂavone contents in soybean during various stages of
germination in mg/ g ground seed, dry basis ....................................... 23

Table 1.3 Potential clinic targets of probiotic intervention ................................... 31

Table 2.1 Analysis of variance for the main factors (independent variables)
on pH of lactic acid bacteria and L. acidophilus NCFM after 6h of
incubation ............................................................................... 49

Table 2.2 Differences in pH, % titratable acidity and growth in blends of
reconstituted 12% non-fat dry milk and germinated/non-germinated soy
powder after 6h incubation ........................................................... 51

Table 2.3 Difference in pH between germinated and non-germinated soybean
varieties ................................................................................. 58

Table 2.4 Analysis of variance for the main factors (independent variables)
on titratable acidity (%TA) of lactic acid bacteria and L. acidophilus
NCFM after 6h of incubation ........................................................ 59

Table 2.5 Difference in percent titratable acidity (%TA) between germinated and
non-germinated soybean varieties ................................................... 60

Table 2.6 Analysis of variance for the main factors (independent variables) on the
growth (log CFU/ml) of lactic acid bacteria and L. acidophilus

NCFM after 6h f incubation .......................................................... 68
Table 2.7 Difference in growth (CPU/m1) between germinated and non-germinated

soybean varieties ....................................................................... 75
Table 3.1 Low-fat whole soy fortiﬁed yogurt formulation ................................... 81

Table 3.2 Overall acceptability of the yogurt samples as determined by untrained
consumer panel (n = 112) ............................................................ 85

Table 3.3 Mean pH of yogurt samples at the time of manufacturing and at the time
of sensory evaluation .................................................................. 87

Table 3.4 Compositional analysis (%) of germinated and non-germinated
soy-fortiﬁed yogurts after manufacturing ........................................... 89

xi

Table 4.1 Nutrient composition (%) of germinated and non-germinated soy
powders .............................................................................. 106

Table 4.2 Total isoﬂavone contents in raw and spray dried germinated and non-
gerrninated soybean powder ( ug/ g) .............................................. 108

Table 4.3 Total Genistein and Genistein contents in raw and spray dried
germinated and non-germinated soybean powder (ug/ g) ....................... 1 10

Table 4.4 Total Daidzein and Daidzin contents in raw and spray dried germinated
and non-germinated soybean powder ( ug/ g) ..................................... 112

Table 4.5 Stachyose contents in spray dried germinated and non-germinated
soybean powder (mg/ g) .............................................................. 1 18

Table 5.1 Total isoﬂavone concentrations in yogurts fortiﬁed with germinated
or non— germinated soybean powders ( ug/ g) at lst and 6th week
of storage (4°C) ........................................................................ 132

Table 5.2 Conversion and retention of isoﬂavones (4 isomers) during
processing of soy powders into yogurts (%) ...................................... 134

Table 5.3 Genistein concentrations in yogurts fortiﬁed with germinated
or non-germinated soybean powders (pg/g) at lst and 6th week
of storage (4°C) ....................................................................... 136

Table 5.4 Genistin concentrations in yogurts fortiﬁed with germinated
or non-germinated soybean powders ( p. g/ g) at lst and 6th week
of storage (4°C) ........................................................................ 137

Table 5.5 Daidzein concentrations in yogurts fortiﬁed with germinated or
non-germinated soybean powders (ug/ g) at lst and 6th week
of storage (4°C) ....................................................................... 139

Table 5.6 Daidzin concentrations in yogurts fortiﬁed with germinated or
non-germinated soybean powders (ug/ g) at 1st and 6th week
of storage (4°C) ....................................................................... 141

Table 5.7 Stachyose contents of yogurts fortiﬁed with germinated or
non-germinated whole soy powders ............................................... 143

Table 5.8 Percent reduction of stachyose in yogurts manufactured with germinated
And non-germinated soy powders .................................................. 145

xii

Table 6.1 Analysis of variance for the effects of yogurt varieties and storage time
(weeks) on the viability of Lactobacillus delbreuckii subsp.bulgaricus
(CFU/ g) ................................................................................. 153

Table 6.2 Viability of Lactobacillus delbreuckii subsp.bulgaricus (CPU/g)
during six weeks of storage at 4 °C ................................................ 156

Table 6.3 Viability of Streptococcus thermophilus (CFU/ g) during
six weeks of storage at 4 °C ........................................................ 159

Table 6.4 Viability of Lactobacillus acidophilus NCFM (CFU/ g) during
six weeks of storage at 4 °C ......................................................... 163

Table 6.5 Analysis of variance for the effects of yogurt varieties and storage time
(weeks) on pH ......................................................................... 164

Table 6.6 pH of soy-fortiﬁed yogurt samples during prolonged cold storage
at 4 °C .................................................................................. 166

xiii

LIST OF FIGURES

Figure 1.1 Structural formula of Isoﬂavones ................................................... 17
Figure 1.2 The structures of oligosaccharides ................................................. 20

Figure 2.1. Schematic diagram of germinated soy powder (GSP) preparation
(Patent#US7, 067,163 B2) .......................................................... 38

Figure 2.2 Germinated soybeans after steeping in acidiﬁed water .......................... 39

Figure 2.3 Wet dehulling process (A wet-type model BB soybean dehuller,

BAR, N.A, Inc., Seymour IL) ...................................................... 40
Figure 2.4 Wet milling process (Model 150 BMI Stainless Steel Mill, BAR,

N.A. Inc., Seymour, IL) .............................................................. 41
Figure 2.5 First stage homogenization process at 3,000psi (Homogenizer—200,

Cherry Burrel Corp. Chicago, IL) .................................................. 42
Figure 2.6 Second stage homogenization at 12,000psi (Rannie 12.56 VH

Homogenizer, APV Americas, Willington, MA ................................. 43
Figure 2.7 Spray dryer ............................................................................ 44
Figure 2.8 Samples of spray dried whole soy powders ....................................... 45
Figure 2.9 Schematic diagram of the activity study ........................................... 46

Figure 2.10 Change in pH of Lactobacillus delbrueckii subsp. bulgaricus (Lr 78) in
reconstituted non-fat dry milk and germinated/non-germinated
(a) Vinton 81 (b) DF 222 (c) E05276-T ........................................... 52

Figure 2.11 Change in pH of Streptococcus salivarius subsp. thermophilus (St 133)
in reconstituted non-fat dry milk and germinated/non-germinated
(a) Vinton 81 (b) DF 222 (c) E05276-T ........................................... 54

Figure 2.12 Change in pH of Lactobacillus acidophilus (La NCFM)) in
reconstituted non-fat dry milk and germinated/non-genninated
(a) Vinton 81 (b) DF 222 (c) E05276-T ........................................... 56

Figure 2.13 Culture activity of Lactobacillus delbruekii subsp. bulgaricus (Lr 78) in

reconstituted non-fat dry milk and germinated/non—germinated
(a) Vinton 81 (b) DF 222 (c) E05276-T ........................................... 61

xiv

Figure 2.14 Culture activity of Streptococcus salivarius subsp.
thermophilus (St 133) in reconstituted non-fat dry milk and
germinated/non—germinated (a) Vinton 81 (b) DF 222 (c) E05276-
T ....................................................................................... 63

Figure 2.15 Culture activity ofLactobacillus acidophilus (La NCFM) in
reconstituted non-fat dry milk and germinated/non-germinated
(a) Vinton 81 (b) DF 222 (c) E05276T ............................................ 65

Figure 2.16 Growth of Lactobacillus delbruekz'i subsp. bulgaricus (Lr 78) in
reconstituted non-fat dry milk and germinated/non—germinated
(a) Vinton 81 (b) DF 222 (c) E05276-T ............................................ 69

Figure 2.17 Growth of Streptococcus salivarius subsp. thermophilus (St 133) in
reconstituted non-fat dry milk and germinated/non-germinated
(a) Vinton 81 (b) DF 222 (c) E05276-T ............................................ 71

Figure 2.18 Growth ofLactobacillus acidophilus (La NCF M) in
reconstituted non-fat dry milk and germinated/non-germinated

(a)Vinton 81 (b) DF 222 (c) E05276-T ........................................... 73
Figure 3.1 Flow diagram for manufacture of cow’s milk/soymilk yogurt .................. 80
Figure 4.1 Standard curve for pure genistein standard ....................................... 99
Figure 4.2 HPLC chromatogram for pure genistein standard at different

Concentrations ...................................................................... 1 00
Figure 4.3 Flow diagram for isoﬂavone extraction from soybean powders ............. 104

Figure 4.4 Representative HPLC chromatogram of isoﬂavones in germinated
(GV 81), non-germinated (NGV 81) and raw (RV 81) Vinton 81
soybean varieties (a, daidzin; b, genistin; c, daidzein; d, genistein ............ 114

Figure 4.5 Representative HPLC chromatogram of isoﬂavones in germinated (GDF
222), non-germinated (N GDF 222) and raw (RDF 222) DF 222 soybean
varieties (a, daidzin; b, genistin; c, daidzein; d, genistein) .................... 115

Figure 4.6 Representative HPLC chromatogram of isoﬂavones in germinated (GET),
and raw (RET) E05276-T soybean varieties (a, daidzin; b, genistin; c,

daidzein; d, genistein) .............................................................. 116

Figure 4.7 Concentrations of soy isoﬂavone isomers in germinated,
non-germinated and raw soy powder ............................................. 1 17

XV

Figure 5.1 Representative HPLC chromatogram of isoﬂavones in
soy-fortiﬁed yogurts atlSt week (a, daidzin; b, genistin; c,
daidzein; d, genistein) .............................................................. 128

Figure 5.2 Representative HPLC chromatogram of isoﬂavones in
soy-fortiﬁed yogurts at 1St week(a, daidzin; b, genistin; c,
daidzein; d, genistein ................................................................ 129

Figure 5.3 Representative HPLC chromatogram of isoﬂavones in
soy-fortiﬁed yogurts at 6th week (a, daidzin; b, genistin; c, daidzein;
d, genistein) ........................................................................... 130

Figure 5.4 Representative HPLC chromatogram of isoﬂavones in

soy-fortiﬁed yogurts at 6th week (a, daidzin; b, genistin; c, daidzein;
d, genistein) ........................................................................... 131

Figure 5.5 Isoﬂavone concentrations in 1 week (A) and 6 week (B) old
yogurt samples ....................................................................... 142

Figure 6.1 Viability counts of Lactobacillus delbreuckii subsp.bulgaricus (CFU/ g)
from germinated and non-germinated whole soy-fortiﬁed yogurt
samples during 6week storage .................................................... 155

Figure 6.2 Viability counts of Streptococcus thermophlilus (CFU/ g) ﬁ‘om
germinated and non-germinated whole soy-fortiﬁed yogurt samples
during 6 week storage .............................................................. 158

Figure 6.3 Growth and viability counts of Lactobacillus acidophilus NCFM

(CFU/ g) from germinated and non-germinated whole soy-fortiﬁed
yogurt samples during 6-week storage ........................................... 161

xvi

INTRODUCTION

During the past decade, there has been an increased interest in nutrition, medical
and food sciences concerning biologically active compounds or health-promoting
components including peptides, which are hidden in the amino acid sequences of food
proteins. These compounds represent potential functional foods or nutraceuticals for food
and pharmaceutical applications. A nutraceutical is deﬁned as a substance that is a food
or part of a food and provides medical or health beneﬁts, including the prevention and
treatment of disease (Kalra, 2003). There is an increased awareness of the health beneﬁts
associated with soybean consumption and the functional foods market currently generate
a lot of dollars in developed countries like the United States. It has been projected that the
nutraceutical market in the United States will increase to about $20 billion by the year
2010 thus contributing to 10% of the US food market (Sloan, 2002).

Numerous natural foods such as milk, soymilk and their derivatives contain
proteins and other compounds which could be made biologically active to form
compounds like antioxidants, conjugated linoleic acid, opioid peptides, hydrolyzates, and
other biologically active compounds as a result of fermentation by lactic acid bacteria or
enzyme hydrolysis (Meisel and Bockelmann, 1999). Several studies have shown that
there is a lower incidence of chronic diseases such as cardiovascular disease, colon,
breast and prostate cancers, with population that consume soy products regularly
(Erdrnan 2000; McCue and Shetty 2004). In these studies epidemiological association
was established between soybean consumption and improved health. Some of these

health beneﬁts have been attributed to bioactive compounds in soybeans known as

isoﬂavones. Research has shown that these isoﬂavones are more prominent in fermented
soy products. Germination of soybean seeds closely resembles fermentation such that
enzymes inherent in the soybeans can hydrolyze the non-bioavailable compounds into
bioactive compounds and further fermentation during yogurt making will increase their
yield. Yogurt is traditionally made from cows’ milk and is consumed in both developed
and developing countries. Recently there has been an increased desire for innovations in
yogurts such as including a variety of ingredients like new strains of probiotics, ﬁbers,
and other components for fortiﬁcation e. g. soy protein isolate, needed for increased
health beneﬁts, hence the interest in soy-based or soy-fortiﬁed yogurt.

The main goal of this proposal is to test the hypothesis that incorporation of
germinated whole soybean powder into cow milk substrate will produce increased yield
of biologically active compounds in the yogurt mix to meet recommended requirements
for added health claims. Five objectives are being proposed to address this hypothesis:

1. Growth and activity of lactic acid bacteria and a probiotic in reconstituted
germinated whole soy powder (GSP), non-germinated whole soy powder (NGSP)
and non-fat dry milk (NF DM) + GSP or NGSP.

2. Development and properties of yogurt from blends of cow’s milk and whole
soymilk base for consumer acceptance.

3. Production and concentration of genistein, diadzein, genistin, daidzin and
oligosaccharide (stachyose) in germinated and non-germinated soy powder.

4. Effect of processing and reﬁdgerated storage on isoﬂavone and stachyose contents
of yogurt fortiﬁed with non-germinated and germinated (predigested) whole soy

powder.

5. Shelf life studies and viability of whole soy-fortiﬁed yogurts stored at 4 °C.
Overall, I wish to establish the fact that better health beneﬁts could be
conferred to consumers by providing bioactive compounds from both soy and cow’s

milk in form of yogurt.

CHAPTER 1

LITERATURE REVIEW

1.1 COW’S MILK YOGURT
1.1.1 Composition and nutritional value of milk

Generally, milk and dairy products have always been considered as important
components of a balanced diet because they provide a wide range of important nutrients.
Proximate composition of cow’s milk contains about 3.3-4.00% protein, 3.65- 4.35% fat,
82.55-88.0% moisture, 0.77-0.81% ash, and 4.0-4.5% carbohydrate calculated as the
difference from 100% (Yadav and others, 2003). Among the minerals present in cow’s
milk, calcium and phosphorous are the most abundant (122.2 and 76.3mg/ 100g
respectively). Milk proteins consist mostly of casein in conjunction with other minor
proteins. The caseins supply the amino acids and several studies have shown the
bioactive potentials of these compounds. Proteolytic enzymes can hydrolyze caseins
easily therefore milk proteins can produce several bioactive peptides such as opoid
peptides, irnmunostimulating peptides, angiotensin I converting enzyme inhibitors and
antibacterial peptides (Y arnamoto, 1997; Clare and others, 2003).

Milk proteins have been known to be the source of different biologically active
peptides but recently several researchers have isolated bioactive peptides from plant
sources such as campesterol, stimasterol and B-sitosterol (Quliez and others, 2003). The
major physiological attributes of these bioactive compounds from milk include their

ability to act as potential modulators of various regulatory processes in the body

(hormonal), immune modulation, antibacterial and antitumor activities (Meisel and
Bockelmann, 1999).

The carbohydrate content of cow’s milk is exclusively lactose sugar. The
cholesterol content is about 14mg/ 100g and it does not contain any dietary ﬁber.
Different processing, methods including pasteurization do not destroy a great portion of
the nutrients including the vitamins (A, B, D, E, K) in the milk although it is fortiﬁed

with vitamins (e. g. vitamin D) sometimes before consumption (Brenda, 2004).

1.1.2 Nutritional and health benefits of yogurt
1.1.2.1 Deﬁnition and history of yogurt

According to Codex Alirnentarius of 1992, yogurt is deﬁned as a coagulated milk
product resulting from the fermentation into lactic acid from milk sugar lactose by
Lactobacillus bulgaricus and Streptococcus thermophilus (Adolfsson and others, 2004).
Other lactic acid bacteria (LAB) could be combined with the traditional yogurt bacteria.
The entire LAB used for yogurt making is expected to be alive and present in large
amounts in the ﬁnished products. Scientiﬁc evidence has shown that cultured milk was
initially made as far back as 4,500 years ago (Van de Water and others, 1999). The
earliest yogurt was made by wild fermentation by the Bulgars in the 2nd century. It
remained primarily a food for the South, Central and Western Asia, South Eastern Europe
and Central Europe. The observation that frequent consumption of fermented milk
containing Lactobacillus bulgaricus increased health and longevity was only made in the

20th century by Metchnikoff, who claimed that the intake of yogurt decreases the toxic

effect of the putrefactive bacteria in the colon by decreasing their growth (Metchnikoff,
1907; Wollowski and others 2001 ).

Cow’s milk alone lack or contain insufﬁcient amounts of amino acids (e. g.
arginine, isoleucine and glutamic acid) and low molecular weight peptides, therefore
cannot support the growth of probotics (Gomes and others, 1998; Hofrnan and Thonart,
2001; Shah, 2000). Alternatively, milk supplemented with soy protein isolate supported
the growth of these probiotics and this could be as a result of enhanced lactose utilization
and acetic acid production due to the presence of these amino acids in the protein isolates

(Pharn and Shah, 2008).

1.1.2.2 Special ingredients of yogurt

Yogurt base, which is milk, is rich in proteins, several B vitamins and essential
minerals. Yogurt is a rich source of calcium and contains as much fat as the milk it is
made from. Overall the nutrient composition of yogurt is based on the nutrient
composition of the milk from which it is made. Several factors affect the nutritional value
of the ﬁnal product. These factors include changes in milk constituents, species and
strains of bacteria used in fermentation, source and type of milk solids, temperature and
time of fermentation (Adolfsson and others 2004). Yogurt is a good vehicle for many
nutritional ingredients because of its high-refrigerated shelf life, ﬂexibility to add any
particular ingredient or nutrient before or after setting.

Yogurt can be easily fortiﬁed with a range of vitamins, antioxidants, probiotics
and other beneﬁcial nutrients. Most of the yogurt proteins come from nonfat milk, ultra

ﬁltered nonfat milk or whey protein. Other nutritional ingredients added to yogurt include

lycopene, omega-3 eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and
Cle O (Gerdes, 2007). Most of these ingredients have well-recognized health claims.
Recently, some yogurt manufacturers add probiotics, prebiotics and phytosterols to the
yogurt mix (Menu and others, 2008). The prebiotics function as dietary ﬁbers and are
mostly polydextrose. Examples of such prebiotics are inulin and oligofructose (Aryana
and others, 2007;Vasiljevic and others, 2007). Inulin is commonly present in plants such
as artichokes, leeks and garlic and is made up of glucose and fructose chains.
Oligofructose is produced ﬁom enzymatic hydrolysis of inulin or partial enzymatic
hydrolysis of sucrose (transfructosylation by B-ﬁ'uctofuranosidase). Both inulin and
oligofructose provide nutritional and functional beneﬁts in foods. Their ascribed
contributions to health beneﬁts include the ability to selectively promote the growth of
probiotics such as biﬁdobacteria and lactobacillus. thereby improve digestive efﬁciency
and health (Bruno and others, 2002).

In 2005, Popa studied the inﬂuence of sweetener type on growth, activity, and
viability of yogurt cultures. Her ﬁndings showed that yogurt ingredients had no inhibitory
effect on the growth of the cultures used. The results also showed that viability of the
lactic acid bacteria and probiotics used was retained at high percentage (85% and 90%
respectively), except in the yogurt sample sweetened with sourwood honey. Vasiljevic
and others (2007) improved probiotic (Biﬁdobacterium animalis ssp. lactis) viability and
stability by adding B-glucan from cat and barley to yogurt.

An increasing popular ingredient in yogurt is phytosterols. These are sterol
compounds, which occur naturally in plants but have long been used to treat

hypercholesterolemia (Monu and others 2008). Phytosterols have become important

ingredients in functional foods because of perceived health potentials. Due to the ability
of these compounds to block cholesterol absorption, thereby reduce coronary heart
disease, it is recommended that consumption of a low-fat yogurt (0.7% fat) that contains

3 g/day of plant stanols will reduce LDL cholesterol by 13.7% (Noakes and others 2005).

1.1.2.3 Biodefense properties of yogurt

The biodefensive properties of yogurt and fermented milk have been well
described by several researchers (Van de Water and others, 1999:Adolfsson and others,
2004). Numerous protective proteins and peptides found in cow’s milk yogurt with
unique biological activities have been documented. The knowledge of dairy science and
technology has afforded scientists the ability to recognize, recover and maintain these
compounds as bioactive functional ingredients that could improve the quality of foods.
Bioactive peptides such as casokinins or angiotensin-converting enzyme (ACE) I
peptides play a role in reducing blood pressure by inhibiting ACE and blocking the
conversion of angiotensin I to angiotensin II, which is a potent vasoconstrictor (Rogelj
2000). Milk peptides have also been associated with anti-carcinogenic properties. Some
of these peptides, according to previous studies, may retard the development of colon
tumors and tumor precursors by providing biologically active methionine and cysteine.
These amino acids are involved in the cellular methylation and DNA stabilization as well
as cellular synthesis of gluthathione, which plays a crucial role in the defense
mechanisms that protect against cancer (Rogelj 2000). The evidence of a higher content
of amino acids supports the argument that proteins from yogurt are more digestible than

proteins from milk. This could be due to bacterial predigestion of the milk proteins in the

yogurt thus releasing free amino acids such as proline and glycine (Shahani and Chandan,
1979)

Previous studies have shown that yogurt and LAB contribute immensely to
gastrointestinal health and thus supports Metchnicoff’ s theory that yogurt may indeed be
beneﬁcial to health. The beneﬁts of yogurt have been shown in both animal and human
studies. These promising health beneﬁts include potential reduction of lactose
intolerance, constipation, diarrhea] diseases, colon cancer, inﬂammatory bowel disease,
Helicobacter pylori infection and allergies (Adolfsson and others 2004; Van de Water
and others 1999). The potential health beneﬁts of LAB will be fully discussed later under
health beneﬁts of probiotics.

Several investigators have studied the effect of yogurt consumption on the immune
system of animals. Their ﬁndings indicate that there is a great potential on enhancing the
immune system by feeding yogurt to mice in terms of improving the immune signaling
compounds. Tejada-Simon and others (1999) studied the effect of consuming yogurt that
contain L. acidophilus and Biﬁdobacterium by mice and their ability to potentiate
immunoglobulin A responses to cholera toxin in those mice. Their results suggested that
ingestion of yogurt supplemented with L. acidophilus and Biﬁdobacterium increased
mucosal and systemic IgA responses to cholera toxin, unlike the control yogurt, which
contained only the conventional starter cultures, where less IgA was produced.

Ha and others (1999), investigated the potential of yogurt consumption to modulate
the immune system by assaying mucosal and systemic cytokine gene expression in mice.
Again these yogurts were supplemented with or without L. acidophilus and

Biﬁdobacterium. They determined relative mRNA for interferon-y, tumor necrosis factor-

or, interleukin-2, interleukin-4 and interleukin-6 from the spleen, mesenteric lymph nodes
or Peyer’s patches. The results showed that long time feeding of some yogurt led to either
a decreased expression of some cytokine mRNAs or had no effect on the test organs,
suggesting that yogurt formulation is critical since different lactic acid bacteria can
differentially affect basal cytokine expression. In another related study, the spleen and
Peyer’s patch lymphocyte populations in mice fed probiotic-supplimented yogurts were
determined (Pestka and others, 2001). Mice were fed for 14 days, after which ﬂow
cytometry was used to determine the phenotypes of immune cells obtained from the
mentioned organs. Results indicated that there was no difference on the amount of CD8+
(cytotoxic T cells), B220+ (B cells), IgA+, or IgM+ cells due to yogurt ingestion, but
there was a signiﬁcant increase in the percentage of CD4+ (T helper) cells ﬁom the
treatment groups containing Bifdobacterium and L. acidophilus and other probiotic-
supplimented yogurts but not in the control groups i.e. mice that were fed conventional
yogmts. These ﬁndings according to the investigators meant that short term feeding
(2weeks) of conventional or probiotic-supplimented yogurts produce minor effect on
lymphocyte distribution in the systemic or mucosal immune compartments.

In the United States, most of the yogurts sold are low-fat or non-fat varieties,
hence lipid hydrolysis makes little contribution to yogurt products attributes. Research
has shown that yogurt contains more conjugated linoleic acid (CLA), a long-chain
biohydrogenated derivative of linoleic acid than just cow’s milk (Rudel, 1999). CLA has
been reported to have both immunostimulatory and anticarnogenic properties (Whigham
and others, 2000). Due to the lower content of lactose in yogurt, the bioavailability of

minerals such as calcium, magnesium and zinc may be reduced since lactose enhances

10

the absorption of these minerals (Bronner and Pansu 1999). Since yogurt is acidic (low
pH), calcium still exists in the ionic form therefore improves intestinal calcium uptake
(Bronner and Pansu 1999). Bacterial cultures used during yogurt making (fermentation
process), inﬂuence the vitamin content of the ﬁnal product. Some of the LAB strains do
not require vitamin B for growth but instead are capable of synthesizing such vitamins.
As such vitamin losses as a result of processing could be corrected by utilizing such

cultures.

1.2 SOYBEAN PRODUCTS
1.2.1 Composition and nutritional value of soybean and soy products

There is an increased awareness of the health beneﬁts associated with soybean
consumption and the functional foods market currently generate a lot of dollars in
developed countries like the United States (Sloan 2002). The use of soy ingredients is
receiving signiﬁcant attention from the food industries and consumers. Soymilk and their
derivatives contain proteins and other compounds, which could be made biologically
active via processing. Several studies have shown that there is lower incidence of
chronic diseases such as cardiovascular disease, colon, breast, and prostate cancers, with
population that consume soy products regularly (Erdman, 2000; McCue and Shetty,
2004). In these studies epidemiological association was established between soybean

consumption and improved health.

11

1.2.1.1 Chemical composition of soybean

Among the recommended alternatives to animal proteins, soybeans remain the
main choice because it contains nutritionally signiﬁcant quantities of isoﬂavones and
have protein proﬁle similar to eggs or red meat (Beasley and others, 2003). The
acceptability of soybean proteins could be attributed to its being nearly equal in
biological value to casein, very affordable and having good functional properties in food
systems. Soybean usage has been part of most Asian culture both as food and medicine
(Messina 1995). Historical documents by the Chinese suggest that soybeans have been
grown and consumed for thousands of years (11th to 7th century BC). Recent ﬁndings
involving their bioactivity in health maintenance has led to increased usage and
consumption. Soybean is a legume which is rich in phenolic compounds and is widely
consumed worldwide especially in Japan, Korea, China and Indonesia and Samuel
Bowen ﬁrst planted soybeans in the United States in Georgia in 1760s in his plantation.

Soy foods rich in isoﬂavones, proteins and some oligosaccharides have been
reported to be involved with many health beneﬁts to human beings (Wiseman and others
2000). Overall, soybeans contain different nutritional components that promote health
beneﬁts and represent an inexpensive and healthy component of consumer diets. Soybean
based products have always been known to be inferior in sensory characteristics due to
beany and off-ﬂavors (Wu and others, 2005). The whole soy plant including the leaves,
stalk and seeds could be utilized as foods, medicine or animal feed. The protein content
of soybean is 38-40% and it is presumed according to previous study to be the most
important nutrient that determines the qualities of most soy products e. g. soymilk, tofu

etc. (Min and others, 2005). The fat content is about 18% (85% unsaturated fatty acids

12

mainly linoleic and linolenic acids). Due to its high content of unsaturated fatty acids,
soybean and its products are susceptible to oxidation (Penalvo and others, 2004). The
carbohydrate content of soybeans is about 30% (made up of 15% each of insoluble and
soluble carbohydrates). Apart from the high content of phytoeostrogens (isoﬂavones),
soybeans are known to be a good source of anti-nutritional factors such as saponins,
phospholipids, protease inhibitors, phytates and trypsin inhibitors.

There are different varieties of soybeans and they vary in protein and oil contents
as well as ﬂavor, seed coat, cotyledon and helium colors and other physical properties
(Min and others, 2005). Also the growing environment apart from bean varieties can
affect the sensory and physical properties of soybeans, which in turn affect the outcome
of the ﬁnal products made from the beans (Table 1.1).

Soybean seed is typically made up of about 90% cotyledon, 8% seed coat and 2%
hypocotyl axis or germ. The cotyledon and germ are excellent sources of macronutrients.
About 80-90% of isoﬂavone is in the germ (Min and others, 2005). Soybean is
successfully cultivated in hot summer climates with optimum temperature between 20°C

to 30°C (68°F to 86°F) and it takes about 80-120 days between sowing and harvesting.

1.2.1.2 Soybean protein

Soy proteins are used in a variety of foods such as salad dressings, soups, imitation
meats, beverage powders, cheeses, non-dairy creamers, frozen desserts, whip topping,
infant formulas, breads, breakfast cereals, pastas and pet foods. They are also used in

non-food products such as adhesives, asphalts, resins, cleaning materials, cosmetics inks,

13

paints, paper coatings, pesticides/fungicides, plastics, polyesters and textile ﬁbers

(Anderson and wolf, 1995)

Table 1.1 Effects of soybean varieties and growing environments on soybean protein, the

protein and total solid contents of soymilk (Min and others, 2005)

 

 

 

Soybean/ Soy bean Soymilk

variety Protein % Protein % Total Solids %
Location
(Columbus)
OH-l 44.3 3.41 d: 0.13be 6.66 i 0.12b
OH-2 40.9 3.37 3: 0.04b 6.60 :1: 0.09b
OH-3 44.2 3.73 :l: 0.08d 6.75 3: 0.06b
OH-4 42.8 3.59 i 0.05“d 6.67 i 0.05b
OH-S 38.5 3.01 :1: 0.138 6.30 i 0.14a
Location
(Lakeview)
OH-l 40.9 3.19 :t 0.02b 6.80 :I: 0.0511c
OH-2 38.8 2.91 i 0.053 6.56 i 0.073
OH-3 39.7 2.97 :I: 0.10a 6.68 :t 007‘”
OH-4 39.9 2.98 :t 0.128 6.70 i 0.06bc
OH-S 40.3 3.21 a 0.06b 6.74 :I: 0.01Cd

 

 

 

14

Soybean protein belong to the globulin family of seed storage proteins called leguminins

(1 IS ﬁ'action) and vicilins (7S fractions). These fractions are known as glycinin and beta-
conglycirrin in soybeans. Soybeans also contain biologically active proteins such as
enzymes, trypsin inhibitors, hemaglutinins and cysteine proteases (Y agasaki and others,
1997)

The soybean protein could be made available in two main forms i.e. soy protein
isolate and soy protein concentrate. Soy protein isolate contains a minimum of 90%
highly reﬁned or puriﬁed form of soy protein, while the concentrate is about 70% soy
protein and it retains most of the original ﬁbers in the soybeans. As previously
mentioned, the biological value of soy protein isolate is comparable to animal proteins
such as egg, casein etc. although it is lacking in the essential amino acid, methionine
which is a sulfur-containg amino acid. The recent approval of soybean protein extract as
dietary supplement by the FDA has further increased the demand for soy foods (U.S.F DA

1999).

1.2.1.3 Soy isoﬂavones

Soybean is rich in phenolic compounds called isoﬂavones (ﬂavonoids). These
isoﬂavones, which belong to the class of phytoestrogens, occur in reasonably high
amount in soybeans and puriﬁed soy proteins. The lack of basic knowledge on the
bioavailability and metabolism of these isoﬂavones has resulted into a lot of
controversies regarding its health related beneﬁts and adverse effects (Cassidy, 2005).
Dietary phytoestrogens are plant derived and have weak and anti—estrogenic properties.

Isoﬂavones are similar structurally to the human estrogens (Tsangalis and others, 2003)

15

and there are twelve chemical forms found in soybeans and soyfoods with varying
concentration in each product (Figure 1.1). The chemical forms include 3 aglycones
(daidzein, genistein and glycitein), 3 B-glycosides (daidzin, genistin and glycitin), 3
acetylglucosides (6”-O-acetyldaidzin, 6”-O-acetylgenistin and 6”-O-acetylglycitin) and 3
malonylglucosides (6”-O-malonyldaidzin, 6”-O-malonylgenistin and 6”-O-
malonylglycitin (Xu and others, 2002; Preinerstorfer and Sontag, 2004).

Soybeans and non-fermented soy foods contain predominantly the glucoside
conjugates of these isoﬂavones, which make up to 80-95% of the total isoﬂavone
concentration (King and Bignell, 2000). The glucoside conjugates are biologically
inactive large molecule and therefore are not easily absorbed directly into the blood
system. The aglycone forms are rarely found in soybeans and soy foods unless the foods
are fermented (Wang and Murphy, 1994). Fermentation of soybeans has been shown to
increase the aglycones and enhance bioavailability of isoﬂavones (Fukutake and others
1996; lzumi and others 2000; Chien and others 2006). These workers indicated that the
aglycones were absorbed faster and in higher amounts than their glycosides in humans.

In vitro fermentation studies have shown that when isoﬂavone glucosides were
incubated with human fecal samples, they were converted into aglycones by the enzyme
glucosidase produced by the fecal bacteria. (Hou and others 2000; Hur and others 2000;
Setchell and others 2002). These same aglycones were further hydrolyzed into
dihydrodaidzein, dihydrogenistein or equols by anaerobic intestinal bacteria. These
studies show that biotranformation of isoﬂavones in the gut are mostly dependent on the
type of intestinal mieroﬂora, thus suggesting that a change in the microﬂora could lead to

a change in isoﬂavone bioavailability.

16

 

 

Aglyc on (non-glyc aside)

Glycoside

Figure 1.1 Structural formula of Isoﬂavones (Izurni et a1. 2000)

 

R]

an

 

.[Glycoside]
Daidzin

Glycitin

EGenistin
6"-0-malonyldaizin
1:6"-0-malonylglycitin
f6"-O-malonylgenistin
6"-0-acetyldaizin
i6"-0—acetylglycitin

f6"-0-acetylgenistin

 

:[Non-glycoside]
Daidzein
iGlycitein

'gGenistein

 

COCHzCOOH

COCH2COOH

COCHzCOOH

1H

H

H

COCH3

COCH3

COCH3

OCH3

OCH3

 

corn

 

17

Certain lactobacilli and biﬁdobacteria are known to hydrolyze B-glucosides,
although the actual bacteria responsible for the metabolism in the intestine are not yet
known (Choi and others, 1999; J con and others, 2002). Chun and others (2007) studied
the conversion of isoﬂavone glucosides to aglycones in soymilk by fermentation with
LAB. Their results suggest that the rates of conversion from glycosides to aglycones
depend on the species of LAB involved. Since Biﬁdobacterium and Lactobacillus are the
dominant bacteria in the intestines, several workers have studied the production of the
enzyme B-glucosidase by Biﬁdobacterium and its ability to transform isoﬂavones into
bioavailable and bioactive forms (Tsangalis and others, 2002, 2004; Wei and others,
2007). Also the ability of certain commercial probiotic lactic cultures (Lactobacillus
acidophilus L10, 8. lactis B94 and L. casei L26) to equally produce B-glucosidase and
biotransform these isoﬂavones in soymilk was studied (Donkor and Shah, 2008). Their
data showed that all the bacteria produced this enzyme and also hydrolyzed B-glucoside
to aglycones in fermented soymilk thereby suggesting an improved biological function of
such soymilk.

Previous investigations have also shown that supplementation of soymilk with
certain ingredients such as skim milk powder, lactulose, calcium etc, in conjuction with
LAB enhanced the biotransforrnation of glycosides to aglycones (Pham and Shah 2007,
2008; Otieno and Shah 2007). The unprocessed soybeans have about 1.2-4.2mg/ g of total
isoﬂavones but this amount can vary due to seed variety, crop year and growth location
(Griin and others 2001). During processing either into fermented or unferrnented
products, some concentrations of the isoﬂavones are lost and also heat, moisture and B-

glucosidases could change isoﬂavone forms and distribution (Wang and Murphy, 1996;

18

Simonne and others 2000; Griin and others 2001; Murphy and others 2002). Some of
these workers observed that up to 61, 44 and 53% of total isoﬂavones, were lost in the
manufacturing of tempeh, toﬁr and soy isolate respectively.

Overall studies have shown that unfermented soy products contain more
isoﬂavones (total), than fermented soy products. Coward and others (1998) reported that
individual isoﬂavones changed their proﬁles as a result of high heat but the total
isoﬂavone contents still remained the same. According to them, baking and ﬂying release
more of malonyl conjugates, moist heat increases B-glucoside conjugate contents, while
dry heat leads to an increase in the acetyl conjugates. Conversion to the aglycone form
and subsequent loss in total isoﬂavone concentration occurred when the product was
heated excessively during manufacturing. In summary, it can be concluded from these
studies that isoﬂavone content in soy based foods is dependent on the variety of soybeans
used, the storage conditions of the raw materials and products and the processing
conditions used during manufacturing. More data are still needed to fully understand the

chemistry and kinetics of isoﬂavones reaction in a model food system.

I. 2. I . 4 Soy oligosaccharides

Oligosaccharides belonging to the raffrnose family, a-galactosides or
galactooligosaccharides (GOS) are non-digestible carbohydrates that commonly occur in
different foods and legumes such as soybeans (Espinosa-Martos and Ruperez, 2006).
Soybean carbohydrates (oligosaccharides) are made up of about 30% sucrose, 18%
stachyose, 6% rafﬁnose and 22% of other saccharides (fructose, rhamnose, arabinose and

glucose) and they are the second largest components in soybeans. The overall properties

19

of an oligosaccharide are dependent on its chemical structure, molecular weight and
contents of contaminating mono- and disaccharides. They are water-soluble and are
typically 0.3 to 0.6 times as sweet as sucrose (Espinosa-Martos and Ruperez, 2006).
Humans are unable to digest these oligosaccharides because they lack the enzyme a-
galactosidase needed to hydrolyze or digest these carbohydrates. The GOS in soybeans
are called rafﬁnose and stachyose. Rafﬁnose is a trisaccharide containing galactose
linked or- (1-6) to the glucose unit of sucrose while, stachyose is a tetrasaccharide
containing a galactose linked a-(1-6) to the terminal galactose unit of rafﬁnose (Figure
1.2). Intact oligosaccharides are preferentially fermented in the colon of the consumer by
biﬁdobacteria resulting in production of gases such as carbon dioxide, hydrogen, methane
etc. Other compounds produced as a result of fermentation are short chain fatty acids,
which have been associated with prebiotic activity with health beneﬁts (Espinosa-Martos
and Ruperez, 2006). I

Figure 1.2 The structures of oligosaccharides (Connes and others, 2004)
110.01, cu,
HO 0 (/ O
~29 04
OH OH
HO
OH H

¥__V__J

Sucrose

 

\r

Rafﬁnose

V
Stachyose

20

As a result of the associated health beneﬁts of these non-digestible
oligosaccharides (prebiotics), they are now regarded as important functional food
ingredient especially in Japan where the Ministry of health and welfare has approved it as
frmctional foods in 1991. Other uses of soybean oligosaccharides include sweeteners
(oligosaccharide syrup) since it has about 60% of the sweetness of sucrose with 50%
fewer calories. Hence potential applications include beverages, baked goods, confections,

ice cream, desserts and health foods.

1.2. 1.5 Soybean germination

Seed germination or sprouting is a biological process in which the plant
becomes ﬁrlly enzymatically active. This process is known to offer numerous nutritional
and health advantages over non- germinated seeds such as increased nutrient
bioavailability, reduction in cooking time and temperature (Khalil and others, 2006; Bau
and others, 2000). The fact that soybeans are known to contain large amount of
isoﬂavones compared to other seeds does not guarantee their presence in sufficient
amounts in products made from soybeans. Germination of whole soybeans consequently
leads to activation of endogenous enzymes of the soybeans resulting to hydrolysis of soy
protein, isoﬂavones, carbohydrates and other molecules, to release bioactive compounds.
According to Bau and others, (2000) soybean germination leads to substantial increase in
certain biochemical and biologically active components of the beans. Some of the
bioactive compounds include lecithin, phytosterols, saponins and oestrogenic compounds
which increased in concentration as a result of germination. Alternatively germination

leads to decreases in levels of rafﬁnose saccharides (Kuo and others, 1988).

21

Soybean germination is also known to increase the activity of the enzyme or-
galactosidase that is responsible for hydrolyzing signiﬁcant amount of low molecular
weight oligosaccharides primarily, stachyose and rafﬁnose into more digestible
carbohydrates such as sucrose and galactose. The inability to digest rafﬁnose and
stachyose generally is due to lack of a-galactosidase, which leads to a reduction in
metabolizable energy and an increase in ﬂatulence and diarrhea. These oligosaccharides
decreased rapidly during germination thus, ultimately leads to reduction of ﬂatulence-
causing properties of non-germinated soybean foods and increase the potential uses of
soybeans in both developed and developing countries. Studies have also shown that short
period of germination can improve the odor and ﬂavor of soy products as a result of
reduced lipoxygenase activities thereby increase product acceptability in developed world
(Bordingnon and others, 1995). Also germination or sprouting deactivates anti-nutritional
factors such as trypsin inhibitors and phytates and transforms soy into a fully enzyme
active safe health food (Zhu and others, 2005).

There is limited information on the effect of soybean germination on isoﬂavone
contents and composition. Terrence (1991) and Zhu and others (2005) reported that the
total isoﬂavone concentration in soybeans increased around 24 hours of germination, and
then concentration decreased slowly thereafter. The total isoﬂavone content at varying
length of hypocotyls of different varieties of soybean seed during germination is shown
in Table 1.2 (Zhu and others, 2005). According to these workers the higher total
isoﬂavones at 0.5 and 2.5mm hypocotyl length could be as a result of increased induction
of the metabolic pathways of naringenin chalcone and isoliquiritigenin, which are the

precursors of isoﬂavonoids in legumes.

22

Table 1.2 Total isoﬂavone contents in soybean during various stages of germination in

mg/g ground seed, dry basis* (Zhu and others, 2005)

 

Soybean variety

 

 

Seed type Hutcheson Caviness
Dry 2.190b 2.286b
Soaked 2.235“ 2.368b
Germinated (hypocotyl 0.5mm) 2.491a 2.7008
Germinated (hypocotyl 2.5mm) 2.442ab 2.78a
Germinated (hypocotyl 6.5mm) 2.304ab 2.486ab
Nongerminated 2.03 5b 2.1 74b

 

*Values in a column with different superscript letters are signiﬁcantly different (p <0.05)

Overall, germination signiﬁcantly increased the total genistein and its B-glucoside
conjugates, daidzein and its B-glucoside conjugates but had little effect on glycitein and
its B-glucoside conjugates. Apart from isoﬂavone concentration change during
germination, other bioactive compounds such as saponins are found more in germinated
soybeans (J yothi and others, 2007). Soybean protein hydrolysates with antioxidant
properties have been produced by germination (Khalil and others, 2006). In general,
soybean germination offers a rare opportunity of using the whole soybean seed and this
will lead to improved human health, increased soy marketability and decrease in

environmental wastes.

23

1.2.1.6 Soy and health

In 2006, consumer awareness of health beneﬁts of soy products was about 82%
(United Soybean Board, 2006). The health promoting effect of soybean consumption was
initially thought to be due to the protein content of soy alone, but recent studies have also
linked these attributes to the biological activities of a speciﬁc group of phenolic
compounds known as isoﬂavonoids (Y amakoshi and others 2000). Soy-based foods
provide beneﬁts for the consumer due to their anticarcinogenic effect, prevention of
cardiovascular disease, prevention of osteoporosis and reduced allergenicity (Pavaro-
Trindade and others 2001; Messina and Messina 2000). The effect of lowering
cholesterol by soy foods has been attributed to the isoﬂavone contents of the food
although the beneﬁcial effects of individually isolated isoﬂavones on lipid markers of
cardiovascular disease have not been fully established yet (Hall and others 2006). These
isoﬂavones, which belong to the class of phytoestrogens, occur in reasonably high
amount in soybeans and puriﬁed soy proteins. The lack of basic knowledge on the
bioavailability and metabolism of these isoﬂavones has resulted into a lot of
controversies regarding its health related beneﬁts and adverse effects (Cassidy 2005).

People with severe allergies to cow’s milk including infants were treated with soy
based formulas or products. In addition, soy formulas have been used to treat medical
indications such as post diarrhea lactose intolerance, galactosemia and primary lactose
deﬁciency (Badger and others, 2002). Soy has been implicated in cancer prevention in
several studies and different workers have shown that early intake of soy could prevent
cancer that would have developed later in life e. g. breast, colon or prostrate cancer. Due

to concerns about hormone replacement therapy (HRT), many pre- and post menopausal

24

women are looking for natural alternatives to reduce or erase the symptoms of
menopause. Clinical studies have shown the potential roles of soy foods and supplements
to combat these adverse effects of menopause. The hypocholesterolernic effects of soy
proteins have been documented and extensively reported, hence the FDA has
recommended intake of soy protein at 25g per day for health claims in soy foods. This is
as result of research by Bakhit and others (1994), where as little as 25 g of soy protein
were able to lower cholesterol in hypercholesterolemic individuals. Soy meal
replacement study suggests that serum cholesterol, LDL-cholesterol, serum triglycerides
were signiﬁcantly reduced as well as the weights of the subjects when compared to casein.
meal replacement (Anderson and Hoie, 2005).

I Isoﬂavones have effects, which are estrogen receptor-mediated and non-estro gen
receptor-mediated (Patisaul and others 2001; An and others 2001; Setchell and Cassidy,
1999) even though they are regarded as weak estrogens. These compounds bind estrogen
receptors and function as estrogen agonists, antagonists or selective estrogen receptor
modulators. According to these workers, these actions or effects are dependent on tissue
or cell types, isoﬂavone concentration and other conditions like hormonal status, age etc
of the individuals. Some studies show that isoﬂavones play a role in inhibiting
angiogenesis, cell differentiation induction, apoptosis and enhancing healthy
cardiovascular ﬁmction including immune function (Scallet and others, 2003). Recent
studies have indicated that soy isoﬂavones indirectly improved cognitive functions in
women (Lethaby and others, 2007). Based on the studies on the health beneﬁts of soy
proteins and isoﬂavones, the importance of the synergistic effects of all bioactive

components of soy on protection from or prevention of diseases cannot be overlooked.

25

1.2.1. 7 Soy products

Different types of soy foods are now available throughout the world but the most
common is soy beverage (e.g. soymilk) especially in the developed world. Two major
types of soy food exist namely fermented and non-fermented soy foods. Traditional non-
ferrnented soy foods include soymilk, soy sprouts, soy nuts, tofu, soy ﬂour etc. (Golbitz,
1995). Fermented soy foods include tempeh, misc, soy sauces and natto. Increased
interest in cow’s milk yogurt alternative, soy isoﬂavones and probiotics has led to soy
yogurt formulations (Farnworth and others, 2007; Lee and others, 2000; Drake and

Gerard, 2003; Donkor and others 2005).

1.3 PROBIOTICS AND HEALTH

A lot of studies have indicated the potential therapeutic effects of LAB
including the probiotics and yogurt. Some of these effects include immunostimulation.
due primarily to yogurt or LAB-induced changes in the gastrointestinal microecology
(Fiander and others, 2005). Probiotic bacteria are deﬁned as “live microorganisms which
when administered in adequate amounts confer health beneﬁts to the host” (F AO/ WHO,
2002). These probiotics must meet certain requirements viz: (l) have the ability to
colonize the host’s intestine; (2) have the ability to survive and withstand exposure to low
pH and bile acids; (3) have the ability to adhere to intestinal epithelium; (4) be
nonpathogenic and nontoxic; (5) host must be able to beneﬁt from it; (6) must be human-
speciﬁc organisms (except, veterinary probiotics); (7) must be stable during storage

(Isolauri and others 2001). Research over the past two decades has shown that probiotic

26

administration could be used to alleviate gut diseases and also prevent and treat other
forms of diseases such as allergies and immune related diseases (Gill and Guamer, 2004).

Research ﬁndings from experimental animals and mostly short-term human
studies have shown that probiotics such as lactobacilli and biﬁdobacteria have the ability
to modulate a host’s immunity and thus making fermented dairy products with probiotics
very popular due to their health beneﬁts (Shah 2006). Consumption of yogurt or lactic
acid bacteria modulates the production of cytokines that play different roles in regulating
immune functions. For example, the use of fermented foods and cultured milk products
containing live microbes has been in existence a long time ago and is believed recently,
to offer a possible means of controlling allergies. The observation that frequent
consumption of sour milk containing Lactobacillus bulgaricus increased health and
longevity was only made in the 20th century by Metchnikoff, who claimed that the intake
of yogurt decreases the toxic effect of the putrefactive bacteria in the colon by decreasing
their growth (Metchnikoff, 1907; Wollowski et a1. 2001). Probiotics have shown some
potential in thwarting the food-bome infections such as salmonella and E. coli. The
antimicrobial effects of probiotics have extensively been studied and their effects on the
microﬂora of the gut cannot be overernphasized. Some probiotics produce short chain
fatty acids, which contribute to the low pH of the colon, thus inhibiting the growth of
pathogenic microorganisms and favoring the growth of the less virulent microorganisms
(Rolfe 2000). Several workers have shown that the health beneﬁts of these probiotics are
dose dependent.

Researches have shown that generally probiotics do not grow very well in cow‘s

milk, therefore in yogurt samples these microorganisms do not attain high numbers

27

unlike the starter cultures (Champagne and others, 2005; Sodini and others, 2002).
However several studies have shown that soy could be a good substrate for probiotic
grth (Mital and others 1974; Nsofor and others 1992; Scalabrini and others 1998),
although they indicated poor growth for traditional yogurt cultures in soy. These studies
suggest that some selected probiotics could compete in the same soy-based substrate as
yogurt cultures, even though limited information is available on the growth of probiotics
in mixed cultures with yogurt starters in soy substrates. The reason is that most studies
are based on the growth of pure cultures on soy substrates or extracts (Kamaly, 1997;
Hou and others 2000; Desai and others, 2002). Farnworth and others (2007) studied the
growth ofLactobacillus sp and biﬁdobacteria in a soy yogurt formulation. Their data
suggest that probiotic bacteria and the biﬁdobacteria were using different sugars for
metabolism when grown in cow’s milk or soymilk. Previous studies on the growth and
metabolism of selected strains of probiotic bacteria in milk also emphasized the
importance of fermentation time since probiotic strains produced different amounts of
metabolic products at various fermentation times (Ostlie and others, 2003). Possible
interactions between starter cultures and probiotics in fermentation of dairy product
should be taken into account when such products are being manufactured since studies
have shown that probiotics are more inhibitory to LAB than vice versa (Vinderola and
others, 2002).

Recent clinical studies have shown that consumption of probiotic drink containing
L. casei, L. bulgaricus and S. thermophilus could decrease the incidence of antibiotic
associated diarrhea in adults (Hickson and others, 2007). Some workers suggest that

traditional yogurt cultures should be considered as probiotics since these cultures are able

28

to eliminate symptoms of lactose intolerance hence improve digestion which is a health
beneﬁt (Guamer and others, 2005). Questions still exist on whether all yogurt cultures
can exert these health beneﬁts and therefore may require further research. Several factors
that affect the effectiveness of probiotics include inclusion of other potentially bioactive
ingredients, the use of probiotic blends, mixing probiotics and prebiotics, growth and
preservation methods for the probiotic strain(s), concentration of probiotics or prebiotics
used, and method of delivery of probiotics to the consumer (Sanders and others, 2005).
The assertion of these workers is that the mode of delivery of probiotics to humans
inﬂuences the target site of the intended product for example using enteric-coated
capsules may be more protective to stomach acid although the biological activity of the
cultures might be unknown. Acid, bile and heat tolerance of eight free and
microencapsulated strains of probiotic bacteria were studied by Ding and Shah (2007).
Their data suggest that microencapsulated probiotic bacteria survived better than free
bacteria in acidic environment, bile salts, and thirty minutes of heat treatment (65° C).
When the cultures were exposed up to one hour of heat treatment, both the free and
microencapsulated probiotic strains had equal viability losses.

Earlier work by Sheil and others (2004) indicated that oral route for probiotic
delivery might not be essential for anti-inﬂammatory effects and also concluded that
responses of probiotics are not disease speciﬁc. These workers injected L.salivarius 118
subcutaneously into IL-10 KO mice in order to attenuate colitis and suppress collagen-
induced arthritis. There was signiﬁcant decrease in colonic inﬂammatory scores

compared to control animals.

29

In summary, since human health issues are very difficult to study directly,
different end points are usually utilized e. g. blood cholesterol is used is an indicator of
heart disease risk. Another burning issue is that not much is known about the mechanisms
by which these probiotics exert their health beneﬁts and that leads to a lot of general
assumptions that can be misleading. Some doubts still exist on the safety of microbial
food supplements thus more work is needed on safety evaluation. Cases of bacterial
translocation and bacteraemia and sepsis have been reported but are rare
(Isolauri and others, 2004). The potential mechanisms of probiotic intervention are shown
in Table 1.3.

Currently, the Food and Drug Administration (FDA) has not established a formal
regulatory category for functional foods including probiotic cultures (Teitelbaum and
Walker, 2002). More attention is called for to ensure the viability of the cultures, prior to
ingestion. Among the most promising factor that could assure culture viability especially
during the storage of probiotic foods (shelf life) is presence of prebiotics in the food
substrate. A prebiotic is deﬁned as “a nondigestible food ingredient that beneﬁcially
affects the host by selectively stimulating the growth and/or activity of one or a limited
number of bacteria in the colon” (Roberfroid, 2000). The main prebiotic with sufﬁcient
scientic data to show for its efﬁcacy in terms of functional food is inulin. Most European
countries have officially recognized chicory inulin and oligoﬁ'uctose as natural food
ingredient, while in the United States, these compounds have a self-afﬁrmed safe status

(Roberfroid, 2000, 2002).

30

Table 1.3 Potential clinic targets of probiotic intervention (Isolauri and others 2004)

 

Effect

Potential mechanism

Potential risks

 

Nutritional management

of acute diarrhea

Nutritional management

of allergic disease
inﬂammatory bowel

disease

Reducing the risk of

infectious disease

Reducing the risk of
allergic/inﬂammatory

disease

Reduction in the duration of rota-
virus shedding, normalization of

gut permeability and microbiota

Degradation/structural modiﬁcation

Risk related to host and

strain characteristics

Strains with proinﬂam-

of enteral antigens, normalization of matory effects/adverse

the properties of aberrant indigenous

effects on innate imm-

microbiota & of gut barrier functions, nity. Translocation/

local & systemic inﬂammatory respo-

nse, increase in the expression mucins

Increase in IgA-secreting cells against

rotavirus, the expression of mucins

infection

Risk related to host &

strain characteristics

Promotion of gut barrier functions, anti- Directing the microb-

inﬂarnmatory potential, regulation of

iota towards other

the secretion of inﬂammatory mediators adverse outcomes/

& promotion of development of the

immune system

31

directing the immune
responder type to
other adverse

outcomes

Prebiotics are food sources that are chosen preferentially by the probotics and the
body does not produce digestive enzymes to hydrolyze it, therefore they serve as sources
of ﬁber or bulk (Lim and others, 2005). The mechanisms for the beneﬁcial effects of
prebiotics have been speculated to include change in the activity of exogenous
carcinogens by modifying metabolic activities and detoxiﬁcation. Also, prebiotics have
the ability to stimulate the production of butyrate (a short-chain fatty acid), and ability to
stimulate certain cytokines in order to modify immune response (Schley and Field, 2002;
Lirn and others, 2005). Some prebiotics are considered normal constituents of the diet
therefore prudent selection of such diets would be beneﬁcial to health (Cummings and
Macfarlane, 2002).

Despite all the above health beneﬁts of soy products, a lot of inconsistencies and
controversies still abound. Epidemiological data suggest that population that consume
soy regularly over a long period of time, have lower incidence of the chronic diseases
earlier mentioned. The need to understand the role of each bioactive component of soy

cannot be overemphasized in order to appreciate their roles in health care.

32

CHAPTER 2

GROWTH AND ACTIVITY OF LACTIC ACID BACTERIA AND A
PROBIOTIC IN RECONSTITUTED GERMINATED WHOLE SOY POWDER
(GSP), NON-GERMINATED WHOLE SOY POWDER (N GSP) AND NON-FAT

DRYMILK (NFDM) + GSP OR NGSP

2.1 ABSTRACT

There is an increased awareness in the role of dietary supplements and
nutraceuticals (bioactive compounds) as regulatory compounds with hormone-like
functions in the human body. Some natural foods such as cow’s milk, soymilk and their
derivatives could be made biologically active via fermentation or enzyme treatment.
Health beneﬁts of soybeans have been attributed to bioactive compounds in the beans.
Incorporation of these compounds into foods such as yogurt will enhance health beneﬁts.
The objective of this study was to evaluate the growth and activity of three lactic acid
bacteria (LAB) in blends of reconstituted non-fat dry milk (N FDM) and germinated
(GSP) or non-germinated (NGSP) whole soy powder obtained from three soybean
varieties (Vinton 81, DF 222 and E05276—T. Growth (CFU/ml), changes in pH and
titratable acidity (%TA) of Streptococcus salivarius subsp. thermophilus (St 133),
Lactobacillus delbruekii subsp. bulgaricus (Lr 78) and Lactobacillus acidophilus (La
NCFM) grown in 12% reconstituted NFDM, GSP, NGSP and blends (3:7, 1:1, 7:3) of
reconstituted NF DM and soy powder were investigated. All the bacterial strains

exhibited comparable growth and acid production in all milk substrates. There was a

33

signiﬁcant interaction (pg .001) between the milk blends, soybean variety and culture
type, although the blends i.e. NFDM/ soy powder was the most signiﬁcant factor (p5
0.001) that affected the activities and growth of LAB strains. Overall, cultures grown in
most milk blends that contained whole GSP or NGSP gave the lowest pH, highest %TA
and growth (CFU/ml) values. However, highest culture activity and growth were
obtained when the cultures were grown in 1:1 NFDM+GSP orNGSP blend. Cultures
grown in GSP blends produced more acid and growth (pg .001) than its NGSP
counterparts. The signiﬁcance of this study is that partial substitution of cow’s milk with
germinated omon-germinated whole soy powder increased growth and activities of LAB
and probiotic that might lead to increase in bioactive compounds to enhance health

beneﬁts of the consumers.

2.2 INTRODUCTION

Some of the probiotic LAB belonging to biﬁdobacteria and lactobacilli have been
introduced into food products for human consumption so as to enhance health beneﬁts.
Consumption of living bacteria such as Lactobacillus rhamnosus GG, Lactobacillus casei
Shirota, Lactobacillus acidophilus NCFB 1748, NCFM, LA5, or Lactobacillusjohnsonii
LAl were shown to positively affect the health of the consumers (Fonden and others,
2000). A minimum number of viable bacteria are required to exert positive health
beneﬁts after consumption. Several studies done have shown that a daily consumption of
fermented dairy products (about 100g), should contain between 106 CFU/ g to 109 CFU/g
of these cultures (Vanderhoof and Young, 1998; Donnet-Hughes and others, 1999).

Unfortunately, this is not the case with some commercially available products (Rybka

34

and Fleet, 1997). The explanation to this anomaly is that probiotics grow very slowly in
these commercial products because they cannot compete with the traditional starter
cultures. Also, it is observed that the probiotics are unstable during storage. A 3-log cycle
decrease was reported for L. johnsonii (LA 1) in fermented milk, after only two weeks of
storage at 4° C hence the need to modify the milk base so as to support probiotic growth.

Milk is considered to be an unsuitable substrate for growing probiotics in the
presence of other lactic acid bacteria because it does not contain adequate amounts of
amino acids and lower molecular weight peptides (Shah, 2006). Based on the poor
growth of probiotic bacteria in the presence of these starter cultures, studies are being
done to improve their growth on milk bases. Nitrogenous compounds, oxygen
scavengers, oligosacchrides, sugars ﬁrom different sources, milk protein concentrates and
casein hydrolysates have all been used in different studies to maintain growth of
probiotics (Dave and Shah, 1996; Shah 2000; Shin and others, 2000; Sodini and others
2002). The results from these studies indicate that there was better survival and stability
of these probiotics.

Soymilk has been reported to be an adequate medium for growth and metabolism
of lactic acid bacteria (Angeles and Marth, 1971). Several amino acids such as arginine,
isoleucine and glutamic acids are low in milk but are higher in soy protein isolate,
therefore probiotic cultures are expected to grow abundantly in milk supplemented with
soy protein isolate (Gomes and others, 1998). The results obtained by Pham and Shah
(2008) show that fermentation of reconstituted skim milk supplemented with soy protein
isolate enhanced lactose utilization and acetic acid production but reduced lactic acid

production and viable microbial population by the probiotics utilized. The growth rates

35

and changes in pH of two strains of biﬁdobateria namely Biﬁdobacterz‘um longum and B.
biﬁdum were investigated in reconstituted skimmed milk, soy milk and modiﬁed MRS
broth (Kamaly, 1997). His data showed that the biﬁdobacterial strains had more
proteolytic activity in soymilk than in reconstituted skimmed milk but growth and pH
changes were more in reconstituted skimmed milk than in soymilk. This study also
indicated that enrichment of soymilk with lactose, galactose, glucose, yeast extract,
proteose peptone, casitone (pancreatic digest of casein), polypeptone and phytone highly
stimulated the growth and acid production by B. biﬁdum. The objective of this study
therefore, is to investigate the suitability of germinated and non-germinated whole soy
powder blends with NF DM as substrates for growth and acid development by yogurt

starter cultures and L. acidophilus NCFM.

2.3 MATERIALS AND METHODS
2.3.1 Materials
2.3.1.1 Cultures

Streptococcus salivarius subsp. thermophilus (St-133), Lactobacillus delbrueckii
subsp. bulgaricus (Lr-78), ﬁom System Bio-Industries (Waukesha, WI) and
Lactobacillus acidophilus (La NCFM) from Danisco (formerly Rhodia, Madison
Wisconsin) were used. The ﬁrst two organisms are the traditional lactic acid bacteria used
in yogurt manufacturing for acid production and ﬂavor, while the third organism was
selected based on the information strongly supporting its probiotic capabilities (Sanders

and Klaenhammer, 2001; Barrangou and others, 2003).

36

2.3.1.2 Soybean

Soybean varieties utilized in this study were Vinton 81, E05276-T and DF 222. Vinton
81 and DF 222 soybean varieties were procured from Michigan Crop Improvement
Association (Lansing, MI). Vinton 81 variety was chosen because it is locally grown and
documented evidence shows uniformity of genetic traits, seed puriﬁcation, increased
growth yield, competitive growth advantages over some other varieties such as high
protein content, disease resistance and performance. A newly developed variety
E05276-T from the Department of Soil and Crop Sciences, Michigan State University
was also used. This is a high yielding variety with high protein content similar to Vinton
81 and low fat content. DF 222 variety is also grown locally and naturally high in protein

content and functionality.

2.3.2 Germinated and non-germinated whole soy powder preparation

An optimized germination process was utilized (patent #US 7,067,163 B2). Five
kilograms of each soybean variety were washed twice in tap water before soaking. The
soak water was acidiﬁed with citric acid to about pH 4.8 and the beans were steeped for
14 to] 6 hours at 24 to 28°C. The steeped water was drained and the hydrated beans
incubated (germinated) for 18 to 24 hours at 24 to 28°C. At the end of incubation period,
the soybeans were de—acidiﬁed with ﬁve times volume/weight water at 45°C for one hour.
The beans were mechanically wet dehulled and milled. The slurry was gelatinized at
92°C for 15 minutes and cooled to 60°C before homogenization at 3,000psi
(Homogenizer-ZOO, Cherry Burrell Corp. Chicago, IL) and 12,000psi (Rannie 12.56 VH

Homogenizer, APV Americas, Willington, MA). The homogenized whole soymilk was

37

spray dried at an inlet temperature of 400°F and outlet temperature of 160°F and stored in
the cold room prior to further studies (Figures 2.1 to 2.7).

The non-germinated whole soybean preparation was similar to the germinated
process except that the soybeans were not allowed to germinate after steeping in acidiﬁed
water for about 16 hours.

Figure 2.1. Schematic diagram germinated soy powder (GSP) preparation
(Patent#US7, 067,163 BZ)
Wash and rinse twice 5kg 1 w fat soy beans in tap water
Acidify with citric acid to pH 4.8

Steep in distilled water @ 24-28°C for 14-16h

Stir moderately, drain, no rinse (hydrated soy beans)

1

Incubate in plastic totes, slightly ajar (3-5cm) @24-28°C for 24-28h
(Germinated soybeans)

Deacidify with 5x vol/wt of water @ 45°C for 1h)
Randomly measure germinatitIr indices (>80% acceptability)
Mechanically dehull and mill with water
i
Gelatinize @ 92°C for 15 min and cool to 60°C

Homogenize @ 3100 and 12,000 psi

Spray dry (germinated soy powder, GSP)

38

 

Germinated soybeans

Figure 2.2 Germinated soybeans after steeping in acidiﬁed water

39

 

Receptacle Soybeans Water

Figure 2.3 Wet dehulling process (A wet-type model BB soybean dehuller, BAR, N.A,
lnc., Seymour IL

40

 

Soybean slurry

Figure 2.4 Wet milling process (Model 150 BMI Stainless Steel Mill, BAR, N.A. Inc.,
Seymour, IL

41

 

 

Homogenized soybean slurry

Figure 2.5 First stage homogenization process at 3,000psi (Homogenizer-200, Cherry
Burrel Corp. Chicago, IL

42

 

Homogenized soybean slurry

Figure 2.6 Second stage homogenization at 12,000psi (Rannie 12.56 VH Homogenizer,
APV Americas, Willington, MA

43

 

Figure 2.7 Spray dryer

 

Figure 2.8 Samples of spray dried whole soy powders

45

2.3.3 Growth and activity evaluation

Conventional yogurt cultures, namely Lactobacillus delbreuckii subsp. bulgaricus
(Lr 78) and Streptococcus salivarius subsp. thermophilus (St 133), and a probiotic,
Lactobacillus acidophilus (La NCFM), were used. Frozen stock cultures (-80°C) of the
LAB were separately inoculated into pasteurized (80 °C for 30 min.) 12% reconstituted
high-heat NF DM (Michigan Milk Producers Association, Ovid MI), and incubated at
37°C for 24h. Activated mother cultures (obtained aﬁer subculturing twice in the same
medium) at 5% culture levels were each inoculated into 12% reconstituted NFDM, GSP,
NGSP or NFDM + GSP/NGSP blends, and incubated at 37°C for 6h (Figure 2.9).
Figure 2.9 Schematic diagram of the activity study

lnoculatel ml of a frozen stock of bacteria in 10 ml reconstituted NFDM and incubate at
37°C for 24-48h

Transfer 0.5 ml of each this culture into 10 ml fresh medium and incubate at 37°C for
24h (repeat once)

1

Transfer 0.5 ml of each culture into 12% reconstituted NFDM, GSP or NFDM/GSP

blend. Incubate at 37°C and 48°C for 24h (inoculate 8 test tubes per culture per milk
base).

Measure pH and titratable acidity at Oh and at 1h interval for 6h or until pH is about 4.6.
Determine SPC at Oh and at 1h interval for 6h

Determine rate of acid production graphically or by calculation
(Note: Steps 4 and 5 should be repeated 2x)

46

The following blends (ratios) were utilized:

GSP/NGSP N FDM
1 0 (control)
0.7 0.3
0.5 0.5
0.3 0.7
0 1 (control)

The pH and titratable acidity (%TA) were measured at Oh and every hour for 6h for each
sample. Change in pH for each sample was determined by subtracting the ﬁnal pH value
(at 6h) from the initial pH (at 0h). Simultaneously, 9ml of each sample was titrated
against 0.1N sodium hydroxide using phenolphthalein as an indicator to determine %TA.
Standard plate count (SPC) was done, reported as CFU/ml at Oh and up to 6b incubation.
One milliliter of each sample was diluted in 99ml of sterile 0.1% w/v bacto-peptone
(Difco Laboratories, Detroit, M1) to determine viable counts of the LAB strains. The
cultures were plated on De Man, Rogosa, Sharpe (MRS) agar (Difco Laboratories,
Detroit, MI) and incubated at 37°C for 24 to 48h. The colonies were counted using a

Quebec colony counter (Fisher Scientiﬁc, Pittsburgh, PA).

2.3.4 Statistical analysis

Each of the treatments was independently replicated 2 times in a randomized block
experiment. All the data were statistically analyzed using Sigma Stat 3.1 (J andel
Scientiﬁc, San Rafael, CA) and Tukey’s test was used for multiple comparisons where p

< 0.05 was considered statistically signiﬁcant.

47

2.4 RESULTS AND DISCUSSION

There was a statistically signiﬁcant interaction between the NFDM and soy
powder blends, soy powder variety and culture types. Table 2.1 shows the analysis of
variance (ANOVA) for the independent variables (main effects) i.e. medium blends i.e.
milk, soy powder ratio (1 :0; 0720.3; 0520.5; 0320.7: 0:1), bean variety (Vinton 81, DE
222, E05276-T) and culture types (Lr 78, St 133, La NCF M), their two-way and three-
way interactions on ﬁnal pH (i.e. after 6h incubation).

The most signiﬁcant factor according to the F-value obtained in a 3-way ANOVA
that affected the activities and growth of the LAB strains is the blends (i.e. milk ratios; F-
value = 41023.820) at p-value S 0.001. This was followed by the culture variety (F-value
= 4941.238) at p-value 3 0.001 , and bean variety (F-value = 2364.470) at p-value S
0.001. The most statistically signiﬁcant interaction was between the blend and culture
variety (F-value = 10165.008) at p-value 5 0.001.

Table 2.2 and Figures 2.1, 2.2, and 2.3 show the change in pH of Lactobacillus
delbrueckii subsp. bulgaricus (Lr 78), Streptococcus salivarius subsp. thermophilus (St
133) and Lactobacillus acidophilus (La NCFM) in the different medium blends after 6h

incubation.

48

Table 2.1 Analysis of variance for the main factors (independent variables) and

interactions on pH of lactic acid bacteria and L. acidophilus NCFM after 6h of incubation

 

 

Mainlantnrs DF SS MS F P
lMedium (milk) blend 4 19.801 4.950 41023.820 <0.001
2Soy powder 4 1.141 0.285 2364.470 <0.001
3Culture 2 1.192 0.596 4941.238 <0.001
Medium blend x Soy powder 16 1.388 0.0867 718.862 <0.001
Medium blend x Culture 8 9.813 1.227 10165.008 <0.001
Soybean powder x Culture 8 0.267 0.0333 276.300 <0.001
Medium blend x Soybean powder x

Culture 32 1.308 0.0409 33 8.822 <0.001
Residual 75 0.00905 0.000121

Total 149 34.919 0.234

 

 

 

 

 

1Medium blend = NFDM/soy powder ratios
2Soy powder = germinated and non-germinated Vinton 81, DF 222 and E05276-T

3 Culture = Lactobacillus delbrueckii subsp. bulgaricus (Lr 78), Streptococcus salivarius

subsp. thermophilus (St 133) and Lactobacillus acidophilus (La NCFM)

49

It is evident that the cultures grown in all the media that contained soy powder and
NF DM gave the lowest pH values except the control that contained only soy powder.
However the highest activity and growth were obtained when they were grown in the
50:50 NFDM + GSP or NGSP blends (e.g. pH = 4.90 while pH of 0 soy: 100 NFDM =
5.97) irrespective of the cultures or bean variety. This observation was similar to the
study by Pham and Shah (2008) when they fortiﬁed reconstituted skim milk with soy
protein isolate. Their study showed that addition of soy protein isolate in the medium
enhanced acid production and growth of probiotic cultures. This could be as a result of
more bioavailable sugars and peptides in the blend for the microorganisms to metabolize.

The results also showed that the cultures grown in GSP blends signiﬁcantly (p5
0.001) produced more acid overall than in its NGSP counterparts (e. g. pH for germinated
Vinton 81 = 5.41; pH for non-germinated Vinton 81 =5 .51) (Table 2.3). Since the non-
germinated soybeans were soaked for a signiﬁcant amount of time before processing into
powder, it was expected that the endogenous enzymes might have contributed to the
release ofbioavailable nutrients for the growth of the LAB. The lower pH values
observed with the germinated samples is in agreement with previous study on germinated
soybean and its effect on fermentation (Ariahu and others 1999). Sodini and others
(2002) made similar observation in a preliminary study on the effect of enriching milk
base with casein hydrolysate for culture growth. They reported that fermentation time
was reduced in some strains due to this effect. Other workers also discovered that the
change in pH was more in milk supplemented with nutrients such as tryptone than in non-

supplemented milk (Ostlie and others, 2003).

50

Table 2.2 Differences in pH, % titratable acidity and growth in blends of reconstituted

12% non-fat dry milk (N FDM) and germinated or non-germinated soy powder after 6h

 

 

 

 

 

 

incubation.

Growth

Blends (ratios) pH %TA (CFU/gnl)
x10

0-0 Soy: 1-0 NFDM 597°" 041° 1.89d
0.3 Soy: 0-7 NFDM 5.68d 068° 1.97°d
0.5 Soy: 0.5 NFDM 4908‘ 060b 281°
0-7 Soy: 0.3 NFDM 5.29b 058° 2.21b
1-0 Soy: 0-0 NFDM 557° 0.47d 204°

 

 

 

 

 

* Different letters column wise denote signiﬁcant difference at p < 0.001;n = 2

51

 

Figure 2.10 Change in pH of Lactobacillus delbrueckii subsp. bulgaricus (Lr 78)

in reconstituted non-fat dry milk and germinated/non-germinated (a) Vinton 81

(b) DF 222 (c) E05276-T

Change in pH

Change in pH

 

 

 

  
 
 
 
 
 
   

 

 

 

2 5 - —o——— NFDM Cont
———o—— 100% NFDM
—~-v-—~- 100% GV81
—w—A— - 100%NGV81 . o
2 0 _ -— -I — 70%NF+30%GV81
— -D -- 70%NF+30%NGV81
—O— 50%NF+50%GV81 ° --X
—o— 50%NF+50%NGV81 .,...:;.-o-‘-'
- ------ A ------- 30%NF+70%GV81 3:7".-
1.5 - ------- v ------- 30%NF+70%NGV81 .°
0 f
e /
1.0 - ,e
c" % v
s /
/ ./ A
o / /
0.5 1 .5 i’ ’X/ .
":30 . "
/ 'ﬂgm.’ ’e’ - —A’
g. ' u . /
0 0 g'u"... o ‘
O 1 2 3 4 5 6 7
Time (h)
2.5 - —o— NFDM Cont
——o—— 100% NFDM
—---v—--- 100% GDF
—--—A— - 100% NGDF
2 0 _ — -§ — 70%NF+30%GDF
' — -1:1 — 70%NF+30%NGDF .‘
——-<>— 50%NF+50%NGDF .°
....... ‘....... 30%NF"70%GDF A...
1.5 30%NF+70%NGDF
1.0
0.5
—c
0.0 a .
0 1 2 3 4 5 6 7
Tim e (h)

52

Figure 2.10 continued.

Change in pH

2.5

2.0

 

NFDM Cont
100% NFDM
100% GET
70%NF+30%GET
50%NF+50%GET
30%NF+70%GET

 

 

53

Figure 2.11 Change in pH of Streptococcus salivarius subsp. thermophilus (St

133) in reconstituted non-fat dry milk and germinated/non-germinated (a) Vinton
81 (b) DF 222 (c) E05276-T

Change in pH

Change in pH

2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0

 

 

NFDM Cont

100% NFDM
100% GV81

100% NGV81
70%NF+30%GV81
70%NF+30%NGV81
50%NF+50%GV81
50%NF+50%NGV81
30%NF+70%GV81
30%NF+70%NGV8

 
 
 
 
 
 
 
   

 

-;
Q3 3
Time (h)
__.__. NFDMCONT
——o— 100%NFDM
_. .7_--- 100%GSMDF222
_. _¢— - 100%NGSMDF222 /'
_ .. — 70%NF+30%GDF /‘
_ .0 — 70%NF+30%NGDF /‘
__.._ 50%NF+50%GDF , ’- ’
___o.— 50%NF+50%NGDF V

30%NF+70%GDF /
30%NF+TO%NGDF

 

 

54

Figure 2.11 continued.

Change in pH

2.0
1.8
1.6 -
1.4

 

NFDM Cont

100% NFDM
100% GSM ET
70%NF+30%G-ET
50%NF+50%G-ET
30%NF+70%G-ET

 

55

 

d

Figure 2.12 Change in pH of Lactobacillus acidophilus (La NCFM)) in
reconstituted non-fat dry milk and germinated/non-germinated (a) Vinton 81 (b)

DF 222 (c) E05276-T

Change in pH

Change in pH

2.0
1.5
1.0
0.5

0.0

1.8
l
1.6 .
1.4 .
1.2 -

1.01

 

 

 

——o— NFDM Cont
—o— 100% NFDM '
— -v— - 100% over /
—. —¢— - 100% NGV81 / f
— -. — 70%NF+30%GV81 /
— -o — 70%NF+30%NGV81 /
—o— 50%NF+50%GV81 / . °
—<>— 50%NF+50%NGV81 .
------- Ann-- 30%NF+70%GV81 ,
....... v....... 30%NF+70%NGV81 / ...A
tel: ..... v
/.I (0'4... . a.
.9'7. K / A
0 .o /
/ /- / .
. n' . / -.. / /
...... . .o"“% I ‘— =
. ""8/ ' 7 /
.Ar-::==-' .4 -
. v ' / - a
. -3 ﬂ};
0 1 2 3 4 5 6 7
Time (h)
——0— NFDM Cont 7
—o— 100% NFDM /
_._,_._ 100%GDF .
— —A— - 100% NGDF /
— -I — 70%NF+30%GDF /-
— -c1 — 70%NF+30%NGDF >
—o—— 50%NF+50%GDF // -'
—<>— 50%NF+50%NGDF A/f
....... An..." 30%NF+70%GDF /

30%NF+70%NGDF ” ‘

 

 

S6

Figure 2.12 continued.

C
1.6 -
___.__ NFDM Cont
- _ -—Q — - 100% GET '
_ .4 — ~- 70%NF+30%GET /’
12 _ _ .4, — 50%NF+50%GET /’
— 4:1 — 30%NF+70%GET /

Change in pH

 

 

 

57

Table 2.3 Difference in pH of cultures between germinated and non-germinated soybean

 

 

 

 

varieties
Soybean Variety Germinated Non-germinated
DF 222 538°” 562°
Vinton 81 541° 5.51b
E05276-T 5,52b NA'

 

 

 

 

 

* =Not Available
** = Different letters denote signiﬁcant difference at pS 0.001.

Table 2.4 shows the analysis of variance (ANOVA) for the main effects i.e.
medium blends, soy powder varieties (V inton 81, DF 222, E05276-T) and culture types
(Lr 78, St 133, La NCF M), their two-way and three-way interactions on ﬁnal titratable
acidity i.e. %TA after 6 hours of incubation. The F -values showed that medium blend is
the most signiﬁcant factor among all the independent variables (F = 8663.034) at p-value
S 0.001. Similarly, the most statistically signiﬁcant interaction was between the medium
blend and culture variety with an F -value of 7592.552 (p S 0.001). This trend is similar to
the results observed with pH results. According to the data obtained, the cultures grown
in medium blends gave the highest %TA (Table 2.4), however the 05:05 and 0.3 :0.7
NFDM + GSP/NGSP blends produced the highest %TA (0.600 and 0.675 respectively).

Meanwhile, the sample with 0 soy: 1.0 NFDM had the lowest %TA of 0.41 0.

58

Table 2.4 Analysis of variance for the main factors (independent variables) and

interactions on titratable acidity (%TA) of lactic acid bacteria and L. acidophilus NCFM

after 6h of incubation

 

 

Mainjasrm DF ss MS F P
‘Medium Blend 4 1.340 0.335 8663.034 <0.001
2Soy powder 4 0.0802 0.0200 518.422 <0.001
3Culture 2 1.177 0.0886 2292.207 <0.001
Medium Blend x Soy powder 16 0.0891 0.00557 144.047 <0.001
Medium Blend x Culture 8 2.349 0.294 7592.552 <0.001
Soy powder x Culture 8 0.0218 0.00273 70.569 <0.001
Medium Blend x Soy powder x

Culture 32 0.0588 0.00184 47.530 <0.001
Residual 75 0.00290 0.000039

Total 149 4.1 19 0.0276

 

 

 

 

 

1Medium blend = NFDM/soy powder ratios
2Soy powder = germinated and non-germinated Vinton 81 , DF 222 and E05276-T

3Culture = Lactobacillus delbrueckii subsp. bulgaricus (Lr 78), Streptococcus salivarius

subsp. thermophilus (St 133) and Lactobacillus acidophilus (La NCFM)

59

Figures 2.13, 2.14, and 2.15 show the rate of acid production (%TA) of
Lactobacillus delbrueckii subsp. bulgaricus (Lr 78), Streptococcus salivarius subsp.
thermophilus (St 133) and Lactobacillus acidophilus (La NCFM) in the medium blends.
According to the results obtained, cultures grown in most of the blends that contained soy
powder gave the highest TA values. However highest activity and growth were obtained
when they were grown in the 05:05 NFDM + GSP/NGSP and 07:03 NFDM +
GSP/NGSP blends (%TA = 0.6 and 0.68 respectively while pH of 0.0 soy: 1.0 NFDM =
0.41) irrespective of the culture type or soy powder variety (Table 2.2). The mean TA
values among bean variety varied from 0.52 for non-germinated DF 222 to 0.58 for
germinated DF 222 (Table 2.5).

Table 2.5. Difference in percent titratable acidity (%TA) of cultures between germinated

and non-germinated soybean varieties

 

 

 

 

Soybean Variety Germinated Non-germinated
DF 222 0,533" 0.52d
Vinton 81 056" 053°
E05276-T 0.56b NA'

 

 

 

 

 

* =Not Available
** = Different letters denote signiﬁcant difference at p5 0.001

60

Figure 2.13 Culture activity of Lactobacillus delbruekii subsp. bulgaricus (Lr 78) in

reconstituted non-fat dry milk and germinated/non-germinated (a) Vinton 81 (b) DF 222
(c) E05276-T

% TA

°/o TA

1.0

0.8

0.6

0.4

0.2

0.0

 

—-—O—- NFDM Cont

—O— 100% NFDM

-‘ -'V-—< - 100% GV81

— —A-—- - 100%NGV81

— -I - 70%NF+30%GV81

— -CI — 70%NF+30%NGV81

—Q— 50%NF+50%GV81

—0— 50%NF+50%NGV81

....... ‘n..... 30%NF+70%GV81
V ------- 30%NF+70%NGV81

  
 
 
 
 
   

I.'
D.
o
.0

 

 

 

 

 

a ‘ = 3 f————-——-+—'—.
0 1 2 3 4 5 6 7
Time (h)

—-.——— NFDM Cont
——O— 100% NFDM

— —' —' - 100% GDF

— —¢— - 100% NGDF

-— -. — 70%NF+30°/oGDF
-- -D -— 70°/oNF+30°/oNGDF
'—.— 50%NF+50%GDF

  
 
 
  
 
 
 

—0—-— 50%NF+50%NGDF A
....... ‘....... 30%NF+70%GDF ...V
....... V"'°°" 30°/oNF+70%NGDF .-

D
O
c'.
.....
0’ o'
.0 .o

 

 

 

410
.l

61

Figure 2.13 continued.

1.2

1.0

%TA

 

NFDM Cont
100% NFDM
100% GET
70%NF+30%GET
50%NF+50%GET
30%NF+70%GET

 

62

 

Figure 2.14 Culture activity of Streptococcus salivarius subsp. thermophilus (St

133) in reconstituted non-fat dry milk and germinated/non-germinated (a) Vinton
81 (b) DF 222 (c) E05276-T

% TA

% TA

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0.8

0.6

0.4

    
 

 

 

 

a
‘ —-—+———— NFDMCONT
—o— 100%NFDM
— —v— - 100% GV81 ’
‘ — 43— - 100% NGV81 .1.
—-.— 70%NF+30%GV81 . ,4“
—-c1— 70%NF+30%NGV81 /
‘ —o—- 50%NF+50%GV81 , ..
—<>-— 50%NF+50%NGV81.-"/’?'/D
....... ‘ooooooo 30%NF+70°/°GV81 . ..0./
-. ....... V ....... 30°/0NF+70%NGV8 '3' .
. .y/‘f-/ _/
/v A
,__,: - v—o "Pi ,A-r "
’V — —A'_ —
.a‘ "
o ’ ._.-¢-"
0 1 2 3 4 5 6 7
Time (h)
- ——o— NFDMCONT
———O— 100%NFDM
— —V— - 100%GSMDF222
-— —A— '- 100%NGSMDF222
— - — 70%NF+30%GDF o
q — -o — 70%NF+30%NGDF -
—O—— 50°/ NF+50%GDF '
° . 4’43
-—0—— 50%NF+50%NGDF / ,/ .
....... .m.... 30%NF+70%GDF [/E/V
....... gnu... 30°/oNF+70°/oNGDF ’. x; ..'
.-/ . ﬁ. . V A
V/ / I ........
..... _— /
O, I..-' v 3
I

 

"—0
#A—‘v I au‘v
with .2 /
v ’ .”
/
,/
0 1 2 3 4 5 6 7

63

Figure 2.14 continued.

C
0.8 -
-—o——— NFDMCONT
———o— 100%NFDM
— -v—» - 100% GSM ET
_ -—A —. - 70%NF+30%G-ET
0.6 - — -u — 50%NF+50%G-ET

— ~13 — 30%NF+70%G-ET

°/o TA

 

 

64

 

Figure 2.15 Culture activity ofLactobacilIus acidophilus (La NCFM) in reconstituted
non-fat dry milk and germinated/non-germinated (a) Vinton 81 (b) DF 222 (c) E05276-T

 

 

 

 

a
0.8 -
——-.-——- NFDM Cont
—-——O—— 100% NFDM v
— --v— .- 100% GV81 ./
— —A— ,_ 100% NGV81 o
0 6 _ —-— 70%NF+30%GV81 /
' — -c1 — 70%NF+30%NGV81 /
—0—— 50%NF+50%GV81 ' ,/A
—o— 50%NF+50%NGV810/ ’ g
....... ‘....... 30°/oNF+70%GV81 .. ..._.,.- v
< ....... V ....... 30%NF+70%NGV81 . ¢ 1: v ;; .........
l— 0.4 T :r n""' 0” I
5° 2..- -—1=
gar-1'?" . -—-. I
a — —¢_‘ =
0.2
0.0 I l T l I I I 1
0 1 2 3 4 5 6 7
Time (h)
0.8 7
——o— NFDM Cont
——o— 100% NFDM
—--—v—~— 100% cor: _.V
-—--—A—--- 100% NGDF /
0 6 - —-.— 70%NF+30%GDF -' ,
' — -D — 70%NF+30%NGDF ./
—o— 50%NF+50%GDF
—-<>-— 50%NF+50%NGDF _/A
....... Ann... 30%NF+70%GDF .. ° ”5;"...
< ....... V ....... 30%NF+70%NGDF ./"—' ’w’ .. --
I: 0'4 - -'/’ . .-- ' 4” '
O\ A , .00 V'.-'? ’-
____—-—” ' ‘- '
ﬁ"w‘;"“"v ”I. J" "’ '
v “naygﬁ ..”_".::. a "
0.2
0.0 T T 1 I I l I |
0 1 2 3 4 5 6 7
Tim e (h)

65

Figure 2.15 continued.

0.7 -

0.6 ~

0.5 ~

 

% TA

 

NFDM Cont
100% NFDM
100% GET
70°/oNF+30°/oGET
50%NF+50%GET
30°/oNF+70°/oGET

 

66

 

Table 2.6 shows the ANOVA for the independent variables and their various
interactions on the growth ofLactobacilIus delbrueckii subsp. bulgaricus (Lr 78),
Streptococcus salivarius subsp. thermophilus (St 133) and Lactobacillus acidophilus (La
NCF M) in the medium blends. Consistent with the results of pH and titratable acidity,
medium blend was the most signiﬁcant factor with an F—value of 112.735 (p< 0.001).
Unlike pH and %TA, soybean powder variety (F-value = 16.028; p< 0.001) had more
signiﬁcant effect on growth than culture type (F-value = 4.967; p= 0.009), although the
interaction between medium blend and culture variety was most signiﬁcant which is
consistent with the other parameters measured. Cultures grown in fortiﬁed NFDM had
highest growth (CPU/ml). The results showed that all the cultures used in this study
successfully attained a desired level, achieving about 108 CFU/ml of each strain in each
blend (Table 2.2; Figures 2.16, 2.17 and 2.18). A concentration of at least 106 CFU/ml
viable cells in product are needed in order to exert health beneﬁts (Ostlie and others,
2003). The 05:05 NFDM and GSP/NGSP blend overall had the highest grth (2.81 x

108 CFU/ml).

67

Table 2.6 Analysis of variance for the main factors (independent variables) and

interactions on the growth (log CF U/ml) of lactic acid bacteria and L. acidophilus NCF M

after 6b of incubation

 

 

Mainjacmrs DF 85 Ms E P
'Medium Blend 4 1.648 0.335 112.735 <0.001
2Soy powder 4 0.0802 0.0200 16.0287 <0.001
3Culture Variety 2 1.177 0.0886 4.96 <0.009
Medium Blend x Soy powder 16 0.0891 0.00557 2.793 <0.001
Medium Blend x Culture 8 2.349 0.294 12.871 <0.001
Bean Variety x Culture Variety 8 0.0218 0.00273 1 7.102 <0.001
Medium Blend x Bean Variety x

Culture variety 32 0.0588 0.00184 3.142 <0.001
Residual 75 0.00290 0.000039

Total 149 4.1 19 0.0276

 

 

 

 

 

Similar to the pH and %TA data, the germinated soybean varieties supported
the growth of the cultures better than their non-germinated counterparts (Table 2.7). This
result substantiates the fact that germination induced a substantial increase in the amount

of bioactive compounds as well as increase in enzyme activities (Bau and others, 2000),

thus providing more bioavailable nutrients for more acid and growth.

68

Figure 2.16 Growth ofLactobacilIus delbruekii subsp. bulgaricus (Lr 78) in
reconstituted non-fat dry milk and germinated/non-germinated (a) Vinton 81 (b) DF 222

(0) E0527

Log CFU/ml

LogCFU/ml

 

 

  
 

 

   
 

 

a
1e+9
1e+8
4.
1e 7 NFDM Cont
—o— 100% NFDM
— _,._. - 100% GV81
— —a— .- 100%NGV81
— - — 70%NF+30%GV81
1e+6 — -c1 — 70%NF+30%NGV81
—o— 50%NF+50%NGV81
....... ‘....... 30%NF+70%GV81
....... V0000... 30%NF+70%NGV81
1e+5 I I I
4 6 8
Time (h)
1e+9 1
1e+8 r
NFDMCONT
1e+7 ' —o——— 100%NFDM
— —v — - 100%GSMDF222
— —A — - 100%NGSMDF222
— -. — 70%NF+30%GDF
— -D — 70%NF+30%NGDF
1e+6 4 —o— 50%NF+50%GDF
—<>— 50%NF+50%NGDF
....... ‘....... 300/0NF+70°/°GDF
....... Vu..... 30%NF+70%NGDF
1e+5 1 I r 1
0 4 6 8

69

Figure 2.16 continued.

  
 

 

 

C
1e+9 -
/ .-
1e+8 -
E
\
3 NFDM Cont
LL 1e+7 . ——o— 100% NFDM
0 — —v-—t - 100%GET
a, — _A,_. - 70%NF+30%GEI'
o — - — 50%NF+50%GH
—‘ — -D — 30%NF+70%GET
1e+6 -
1e+5 . t t .
0 2 4 6
Time (h)

70

Figure 2.17 Growth of Streptococcus salivarius subsp. thermophilus (St 133) in

reconstituted non-fat dry milk and germinated/non-germinated (a) Vinton 81 (b)

DF 222 (c) E05276-T

Log CFU/ml

LogCFU/ml

1e+9 -

1e+8 .

1e+7 ~

1e+6 -

  
 

 

1e+5

NFDMCONT
—o— 100%NFDM
— ._.,._. - 100% GV81
— -—A—« - 100% NGV81

_ —. — 70%NF+30%GV81
_ -D — 70%NF+30%NGV81
—+— 50%NF+50%GV81
——-()-— 50%NF+50%NGV81
....... 30%NF+70%GV81
------ 30%NF+70°/0NGV8

 

1e+9 -

1e+8 -

1e+7 -

1e+6 -

 

1e+5

 

A
O)
on

NFDMCONT
100%NFDM
100%GSMDF222
100%NGSMDF222
70%NF+30%GDF
70%NF+30%NGDF
50%NF+50%GDF
50%NF+50%NGDF
30%NF+70°/oGDF
30%NF+70%NGDF

 

71

Figure 2.17 continued.

  
 

 

 

C
1e+9 -
1e+8 -
E
\
E
O 1e+7 - NFDMCONT
c) ———o————— 100%NFDM
o - -v~— - 100% GSM er
_1 — -A‘— - 70%NF+30%G-ET
— -u — 50%NF+50%G-ET
1e+6 . — -D — 30%NF+70%G-ET
1e+5 1 r t .
2 4 6
Time (h)

72

Figure 2.18 Growth ofLactobacilIus acidophilus (La NCF M) in reconstituted
non-fat dry milk and germinated/non-germinated (a) Vinton 81 (b) DF 222 (c)
E05276-T

1e+9 -

1e+8 .

  
 
  
 

NFDM Cont
—-O-—- 100% NFDM
—'-'—‘- 100%GV81
—- wA — - 100% NGV81

— -. — 70%NF+30%GV81
—- ~D -- 70%NF+30%NGV81
—.—— 50%NF+50%GV81
—0— 50%NF+50%NGV81

Log CFU/ml
a;
r,

 

 

 

 

+ d
16 6 ....... ‘ ....... 30%NF+70%GV81
....... v------- 30%NF+70%NGV81
1e+5 t I ' I F
0 2 4 6 8
Time (h)
1e+9 -
1e+8 -
E
3
LL 1e+7 4 NFDMCONT
0 ——o—— 100%NFDM
8) _. —v— - 100%GSMDF222
_I -—4 %'—‘ - 100%NGSMDF222
— -. — 70°/oNF+30°/I)GDF
1 +6 - _ .0 _ 70%NF+30%NGDF
e ___..—. 5o%NF+50%GDF
‘ : —<>— 50%NF+50%NGDF
.-° ......... ‘ ....... 30%NF+70%GDF
A' ....... q ....... 30%NF+70%NGDF
1e+5 t a T ' 1
o 2 4 6 3

73

Figure 2.18 continued.

 

 

 

C
1e+9 l
1e+8 ‘
E
\
E
0 19+7 '
U)
3 NFDMOONT
100%NFDM
100% GSM ET
“3+6 ‘ 70%NF+30%G-EI'
50%NF+50%G-El'
30%NF+70%G—El’
1 6+5 1 1 l r I
0 2 4 6 8
Time (h)

74

Table 2.7 Difference in growth (CFU/ml) of cultures between germinated and non-

germinated soybean varieties

 

Bean Variety Germinated (x108) Non-germinated (x108)

 

 

 

DF 222 2,393“ 211°
Vinton 81 2.26b 205°
E05276-T 2,100 N/A”

 

 

 

 

 

* =Not Available

** = Different letters denote signiﬁcant difference at p5 0.001

Overall these data strongly support the view of several workers that non-fortiﬁed
milk does not support LAB growth as much as fortiﬁed milk (Kamaly 1997; Pham and
Shah, 2008). Recent work showed that pH drop during the fermentation of soy beverages
was faster by Streptococcus thermophilus (ATCC 4356), Lactobacillus delbruekii subsp.
bulgaricus (1M 025) and certain probiotics such as Lactobacillusjohnsonii NCC533 (LA-
1), Lactobacillus rhamnosus ATCC 53103 (GG), biﬁdobacteria than in cow’s milk
(Farnworth and others 2007). From their research, these workers concluded that
probiotic bacteria utilized different sugars to support growth when grown in either cow’s
milk or soy beverage. Our study indicates that addition of whole soy powder promoted
cell growth and acid production. This work shows that more than 106 CFU/ml needed to
exert health beneﬁts to consumers was obtained when NFDM was fortiﬁed with whole

soy powder.

75

CHAPTER 3

DEVELOPMENT AND PROPERTIES OF YOGURT FROM BLENDS OF
COW’S MILK AND WHOLE SOYMILK BASE FOR CONSUMER

ACCEPTANCE

3.1 ABSTRACT

Consumer awareness of soy as a healthy food has increased substantially in the
past decade, therefore the growth potential has enticed food manufacturers to extend
products by replacing larger quantities of dairy ingredients with soy. 50:50 blends of high
heat non-fat dry milk (N FDM) and germinated (GSP) or non-germinated (N GSP) whole
soy powders were utilized for yogurt manufacturing for preliminary sensory evaluation.
Experienced yogurt consumers screened the fortiﬁed yogurt samples and selected
samples made with soy powders from Vinton 81 and DF 222 soybean varieties as the
most acceptable for pilot plant yogurt production. Six different strawberry ﬂavored
yogurts were manufactured as follows: GSP Vinton 81 + NFDM, NGSP Vinton 81 +
NFDM, GSP DF 222 + NFDM, NGSP DF 222 + NFDM, NGSP Vinton 81- all soy
control and NFDM- all dairy control. A total of 112 untrained panelists evaluated each
sample for appearance, body texture, ﬂavor and overall acceptance on a 9-point hedonic
scale. There was no statistically signiﬁcant difference between 100% NFDM and the

50:50 blended yogurts for ﬂavor and overall acceptance (p=0.0001). Overall the 100%

76

soy yogurt had lower sensory scores than the other yogurts. Also, the pH of the 100% soy
yogurt was signiﬁcantly higher than the other samples (4.67) even though it was still
within acceptable pH range for yogurt.

Nutrient composition analysis indicates that the protein contents of all the yogurt
samples ranged from 4.80% (100% soy yogurt) to 6.82% (50% E05276-T powder + 50%
non-fat dry milk). There was no statistical difference between the protein content of
100% dairy yogurt and the remaining soy-fortiﬁed yogurt samples. On the other hand
100% soy yogurt had 1.32% fat while 100% dairy yogurt had 0% fat since it was
manufactured from non-fat dry milk only. The carbohydrate contents ranged from
13.62% for yogurt fortiﬁed with non-germinated Vinton 81 powder and 19.39% for
yogurt fortiﬁed with germinated E05276-T powder. The ash contents varied from 2.08%
' for 100% soy yogurt to 4.09% for 100% dairy yogurt. Alternatively, the neutral detergent

ﬁber varied ﬁom 0% for 100% dairy yogurt to 1.6% for 100% soy yogurt.

3.2 INTRODUCTION

Traditionally, yogurt is usually made with a base of dairy milk with high protein
content. Food scientists and manufacturers view yogurt as a perfect delivery vehicle for
vitamins, ﬁbers, essential fatty acids, antioxidants, probiotics etc, thus several added
health beneﬁts have been attached to it especially to a speciﬁc population e. g. aged
individuals. The nutritional value of yogurt is dependent on the nutrient content of the
milk it is made from although some minerals are more bioavailable due to fermentation
(Adolfsson and others, 2004). Some vitamins like B-6 and B-12 are decreased while

peptides, free amino acids, free fatty acids, folic acid and choline contents are increased

77

in yogurts (Meydani and Ha, 2000). Addition of phytosterols to foods such as yogurt is
gaining popularity as a result of its ability to reduce serum cholesterol (Hansel and others
2007; Monu and others, 2008). Lactic acid bacteria especially probiotics are used both to
preserve foods as well as promote good health, but strict strain dependence and poor
growth and survival under different processing conditions have resulted in limited use of
cultures and probiotics addition and has increased the use of microencapsulated cultures
leading to extra manufacturing cost (Dave and Shah, 1996; Bruno and others, 2002;

Donkor and others, 2006; Aryana and others 2007).

The therapeutic effects of cow milk yogurt consumption on gut function have been
investigated and reviewed (Van de Water and others 1999; Adolfsson and others 2004).
Substantial evidence supports the beneﬁcial effects of yogurt and probiotics although
there are still some inconsistencies in reported data for the use of probiotics. These
inconsistencies could be attributed to strain differences, routes of administration of
cultures or improper research design. The addition of dietary ﬁbers into foods is
becoming popular. Due to FDA’s approval for soy protein health claim concerning heart
disease, the soy market has doubled. In addition, many studies supporting the ability of
soy to improve or protect against postmenopausal symptoms, osteoporosis,
hyperlipidemia, prostrate enlargement, bladder cancer, hypertension, as well as other
types of cancer have been published (Bager and others 2002). According to USDA
Nutrient data base for Standard Reference, 100g of soybeans provides 36.5 g of protein,
277mg of calcium, 15.7 g of iron, 280mg of magnesium, 704mg of phosphorous, 1797mg
of potassium, 4.9mg of zinc and varying amounts of vitamins A, E, C, B1, B2, B3, B5,

B6 and folic acid. Despite all the claims about soy beneﬁts to health a lot of controversies

78

still exist concerning its safety to consumers such as estrogenicity and increased cancer

risk as a result of high isoﬂavone consumption (Tsangalis and others 2002).

Culture and familiarity play important roles in the perception, description and
acceptance of food products. A study done in France and Vietnam showed that only a
small cultural difference was observed on perception of soy and cow milk yogurts (Tu
and others 2007). Meanwhile in the same study, a signiﬁcant difference was observed in
the two groups in the verbal description of yogurt aroma especially with soy yogurt.
Their investigation simply suggested that globally, the perception of soy yogurt
characteristics is not inﬂuenced by culture but by use of familiar terminology unique to
each culture to describe the same attribute e. g. aroma. Another study on culture-speciﬁc
variation in the ﬂavor proﬁle of soy product concluded that culture-speciﬁc preferences
are the determining factor in ﬂavor proﬁles of soymilk from distinct geographical regions
(Keast and Lau, 2006). Presently, little if any research on the fortiﬁcation of dairy

yogurt with soy protein or whole soy is known.

The aim of this study therefore is development and properties of yogurt from

blends of cow’s milk and whole soymilk base with consumer acceptance.
MATERIALS AND METHODS
3.2.1 Low fat yogurt formulation and manufacture

Swiss-style strawberry ﬂavored low fat yogurt was made using 50:50 blends of all

the soybean powder varieties of germinated (GSP) and non-germinated (N GSP) Vinton

79

81, germinated (GSP), non-germinated (N GSP) DF 222 and germinated (GSP) E05276-T
and NFDM. Figure 3.1 shows the ﬂow diagram for the yogurt manufacturing process.
Figure 3.1 Flow diagram for manufacture of cow’s milk/soymilk yogurt.

Mix milk blends, stabilizer, and sweetener

Homogenize dual stage 2000, 500psi at 60 °C

1

Heat treatment 85 °C, 30 min.
Cool to 43 °C

Add LAB and Probiotic

l

Incubate at 43 °C until pH 4.4-4.6

l

Blend strawberry puree
Package and cool 4 °C and store at 4 °C

Seven percent high heat NF DM (Michigan Milk Producers Association, Ovid, MI)
was blended with 7% (total solids) soy powder, 0.5% stabilizer (Tate and Lyle Custom
Ingredients, Sycamore, IL) and 7% sucrose (Michigan Sugar Company, Saginaw, MI).
Moisture content of the soy powders was taken into consideration and adjustment was
made for each powder to give 7% total solids (Table 3.1). All the yogurt milk bases were

homogenized at 2000 and 500 psi (Homogenizer-200, Cherry Burrell Corp. Chicago, IL)

80

at 60°C and batch pasteurized at 85°C for 30 minutes. The mixed bases were cooled to
43°C and inoculated with 0.5% (w/v) commercial yogurt cultures YC-Xll (Chr. Hansen
Laboratories, Milwaukee, WI), and a probiotic Lactobacillus acidophilus culture NCFM
(Danisco US, Madison, WI). Inoculated yogurt bases were incubated at 43 °C until pH
was about 4.4-4.6. At the end of incubation period 13% strawberry puree (Kraus & Co.,
Walled Lake, MI) was added to each yogurt batch and mixed thoroughly. All the yogurt

samples were packaged into 802 cups and stored at reﬁigeration temperature (4°C) for

further analysis including sensory and shelf life evaluations.

Table 3.1 Low-fat whole soy-fortiﬁed yogurt formulation

 

 

 

 

 

 

 

 

 

N GDF

Ingredients (%) CV 812 NGV 81 GDF 222 222 GET
Soy powder 7.58 7.59 7.58 7.51 7.56
NFDMI 7.00 7.00 7.00 7.00 7.00
Sucrose 7.00 7.00 7.00 7.00 7.00
Stabilizer 0.50 0.50 0.50 0.50 0.50
Strawberry puree 13.00 13.00 13 .00 13.00 13.00
Added water 64.92 64.91 64.92 64.99 64.94
Total 100.00 100.00 100.00 100.00 100.00

 

 

 

 

 

 

1NFDM = Nonfat dry milk

2GV 81 = Germinated Vinton 81 powder
NGV 81 = Non-germinated Vinton 8] powder
GDF 222 = Germinated DF 222

NGDF 222 = Non-germinated DF 222

GET = Germinated E05276-T

81

 

3.2.2 Screening of Swiss-style strawberry ﬂavored low-fat yogurt by experienced
consumers

Experienced yogurt consumers made up of Dairy and Food Processing Professors,
Dairy Plant Manager and Food Technologists and some members of Dairy Products
Evaluation Team screened the yogurt samples made ﬁom 50:50 blends of all the different
soybean powder varieties namely GSP and NGSP Vinton 81, GSP and NGSP DF 222
and GSP E05276-T. An evaluation form was provided for each screener to simply
indicate acceptable (score of l) or unacceptable (score of 0) (Appendix 1) for the
following yogurt attributes: ﬂavor, body and texture, appearance and color, and overall
acceptance. Five varieties of strawberry ﬂavored yogurts were manufactured and
randomly labeled with three-digit number as 252 (GSP Vinton 81 + NFDM), 169 (NGSP
Vinton 81 + NFDM), 344 (GSP DE 222 + NFDM), 159 (NGSP DF 222 + NFDM), and
817 (GSP E05276-T). Samples containing GSP/NGSP Vinton 81 and GSP/NGSP DE
222 varieties were chosen as the most acceptable by the experienced screeners for yogurt
production. These selected samples were then manufactured on a larger scale at the
Michigan State University dairy pilot plant for the sensory evaluation using untrained

consumer panelists.

3.2.3 Sensory evaluation of Swiss-style strawberry ﬂavored low-fat yogurt:
Untrained Panel
The untrained panelists were recruited through e-mails containing the ﬂyers
(Appendix 2) to students (graduate and undergraduate), faculty and staff of different
departments but mostly from Food Science and Human Nutrition Department. Also ﬂyers

were posted around the Food Science Department buildings. Prior to the initial screening

82

of yogurt samples, permission was sought and approved for the use of human subjects for
this study by the University Committee on Research Involving Human Subjects
(UCRII-IS) (Appendix 3). A total of 112 untrained panelists took part in the sensory
evaluation exercise. The evaluation was conducted in booths located in the sensory
laboratory in the Department of Food Science and Human Nutrition at Michigan State
University (MSU). Before participating in the actual testing, the consent form (Appendix
4) was given and explained to each panelist for signing.

Six varieties of strawberry ﬂavored yogurts were manufactured. These were
randomly labeled with three-digit numbers as follows: 252 (GSP Vinton 81 + NFDM),
169 (NGSP Vinton 81 + NFDM), 344 (GSP DF 222 + NFDM), 159 (NGSP DF 222 +
NFDM), 894 (N GSP Vinton 81-all soy control), and 949 (NFDM-all dairy control). The
yogurt samples were scooped into 402 plastic cups and labeled accordingly with the
selected three-digit numbers. Each panelist was presented with six yogurt samples
(maintained at reﬁigeration temperature) in a random order so as to eliminate bias across
different individuals. The panelists were asked to evaluate all six samples for appearance,
body texture, ﬂavor and overall acceptance and indicate their degree of liking on a nine-
point hedonic scale from 1 = dislike extremely to 9 = like extremely; 5 = neither like nor
dislike (Appendix 5). Drinking water was provided for panel members to rinse their pallet

in between samples in order to avoid carry over of taste from one sample to another.
3.2.4 Proximate analysis of yogurt samples

The following parameters were measured namely crude protein, fat and

carbohydrate (determined by subtraction). Also measured were ash, dietary ﬁber and dry

83

matter. All the measurements were carried out in the Dept. of Animal Science, Michigan
State University. Standard or slightly modiﬁed standard methods were employed. Ether
extraction method was used for the crude fat analysis (AOAC 2005), neutral-detergent
ﬁber method was utilized for dietary ﬁber analysis (Goering and Van Soest, 1970;
Robertson and Van Soest, 1977; Chemy and others, 1989). The total nitrogen (modiﬁed
Kjeldahl digestion) method was used for the protein analysis (Hach and others, 1987).
Ash content was determined using the AOAC (2005) method 945.46 and carbohydrate

was estimated by difference.

3.2.5 Statistical analysis

All the data were analyzed using one-way analysis of variance (ANOVA).
Tukey’s test was used for multiple comparisons of the means. SIM 2000 Sensory
Evaluation Soﬂware, version 6.0(Sensory Computer Systems, Morristown, N.Y., USA.)
was used for the sensory evaluation analysis. Sigma Stat 3.1 was utilized for the analysis
of nutrient composition data (replicate). ANOVA data with p < 0.05 were considered as

statistically signiﬁcant.

3.3 RESULTS AND DISCUSSION
3.4.1 Sensory evaluation of yogurt samples

Table 3.2 summarizes the results of the sensory analysis by a consumer panel. The
sensory parameters measured were appearance, body texture, ﬂavor and overall

acceptance.

84

Table 3.2 Overall acceptability of the yogurt samples as determined by untrained

consumer panel (11 = 1 12)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Attribute 252' 169 949 344 159 894 value Signiﬁcance
Appearance 6.80ab 680°b 6.57"b 696°b 701° 624° 0.0001 ***

Body Texture 6.3455 6.07ab 6.51° 625°b 640° 581" 0.064 **

Flavor 543° 521° 558° 539° 567° 4.43b 0.0001 ***
Overall 548° 532° 5.71a 559° 5.72a 4.55b 0.0001 ***
Acceptance

 

3* Means in the same row with different small letter superscripts are signiﬁcantly

different.

Scale: 9 = like extremely; 5 = neither like nor dislike; l = dislike extremely.

1252 = germinated Vinton 81 soy powder + NFDM

169 = non-germinated Vinton 81 soy powder + NFDM
949 = all dairy NF DM-control

344 = germinated DF 222 soy powder + NFDM

159 = non-germinated DF 222 soy powder + NFDM

894 = all non-germinated Vinton 81-control

In this particular study, the treatment effect was associated with the addition of

50% whole soy powder to the yogurt base, while the two controls consisted of either

100% NFDM (sample 949) or 100% whole soy powder (sample 894). The mean score

values for overall acceptance for all the treatment and control samples were between 4.55

and 5.72, which corresponded to “dislike slightly” and “neither like nor dislike”

respectively. Overall, based on the acceptability mean score, there was no statistically

signiﬁcant difference between the 100% all dairy (NFDM) and the 50:50 blended yogurts

85

 

for ﬂavor and overall acceptance (p = 0.0001). Overall consumer preference for NFDM
whole soy-fortiﬁed yogurts was Similar to 100% dairy yogurt. The 100% soy yogurt

(N GSP Vinton 81) scored lower overall in all the sensory attributes measured than the
other yogurts. This could be attributed to the higher ﬁber contents present in the whole
soy powder utilized, which contributed to increased viscosity, and thus thick body (Drake
and others 2000). This observation is also reﬂected in the scores for body texture where
100% soy yogurt (sample 894) had the lowest score (5.81), while 100% NFDM yogurt
(sample 949) had the highest score (6.51) although it was not statistically different from
the soy-fortiﬁed yogurts (p = 0.064) (Table 3.2).

In terms of yogurt appearance, the mean scores ranged from 6.24 for sample 894
(100% soy) to 7.01 for sample 159 (non-germinated DF 222 and NFDM). This simply
means that the subjects “liked slightly to “moderately liked” the appearance of all the
yogurt samples. The data also showed that the consumers in some cases signiﬁcantly
preferred the appearance of the soy-fortiﬁed yogurt to the non-fortiﬁed yogurt. Yogurt
samples made ﬁom DF 222 had higher ratings than samples made from either Vinton 81
or NFDM. This could be as result of the lighter yellow (creamy) color of DF 222 soy
powder as opposed to a more intense yellow color of the Vinton 81 soy powder. Previous
study by Min and others (2005) indicated that soybean varieties and growing locations
could signiﬁcantly affect chemical and physical properties of soybean and soy foods
especially the protein and total solids contents of the corresponding soy foods. The

strawberry puree was responsible for the pink color of the yogurt samples.

86

3.4.2 Effect of whole soy powder fortiﬁcation on pH of yogurts

The pH values of the yogurt samples are shown in Table 3.3. Prior to the sensory
evaluation (a week after manufacturing), the pH values of the yogurt samples were
measured and there was no signiﬁcant difference in pH that was originally measured
immediately after incubation.

Table 3.3 Mean pH of yogurt samples at the time of manufacturing and at the time of

sensory evaluation

 

 

 

 

 

 

 

pH pH
Sample at time of at time of sensory
manufacturing evaluation
I252 4.54 a 001° 4.550 :r 0.01c
169 4.60 1: 001° 4.590 a 0.01c
344 4.40 a 001° 4.390 a 0.01°
159 4.480 a 0.01b 4.490 a 0.01b
894 4.65 a 001° 4.670 a 001°
949 4.41 a 001° 4.405 a 001°

 

 

 

 

 

“1 Different superscript letters denote signiﬁcant difference (p 3 0.0001), n = 2 for all
samples.

1252 = germinated Vinton 81 soy powder + NF DM

169 = non-germinated Vinton 81 soy powder + NFDM

949 = all dairy NF DM-control

344 = germinated DF 222 soy powder + NFDM

159 = non-germinated DF 222 soy powder + NFDM

894 = all non-germinated Vinton 81-control

87

Sample 894 (100% soy yogurt) had the highest pH but overall, all yogurt samples
were within the acceptable range of yogurt pH. Yogurt fortiﬁed with germinated DE 222
had the lowest pH and was not Signiﬁcantly different from pH of 100% dairy yogurt. This
result is consistent with the result obtained in objective 1 on culture activity where
germinated DF 222 had the lowest pH when the cultures were grown in it. The higher pH
values obtained in sample 894 could be attributed to the fact that it has less hydrolyzed

carbohydrates for the lactic acid bacteria to metabolize.

3.4.3 Nutrient composition of yogurt samples
The protein, fat, carbohydrate and ash contents of the soy-fortiﬁed yogurts are
shown in Table 3.4. The results on the proximate analysis indicate that there are
statistically signiﬁcant differences in the mean values (p 5 0.001), of each parameter

measured.

3.4.3.1 Eﬂect of soy fortiﬁcation on protein content of yogurt

The data in this study Show that there was no signiﬁcant difference (p< 0.05) in
protein content between the all-dairy control (sample 949) with 5.56% protein and the
germinated and non-germinated Vinton 81 fortiﬁed yogurts (samples 252, 5.98%protein
and 169, 5.87% protein), respectively. Also the 100% dairy yogurt was not statistically
different (p< 0.05) from germinated DF 222 fortiﬁed yogurt (5.16%) but was different
from non-germinated DF 222 fortiﬁed yogurt (5.08%) and 100% soy yogurt (sample 894,

4.80% protein). Germinated E05276-T fortiﬁed yogurt (sample 817, 6.82% protein) was

88

signiﬁcantly higher (p< 0.05) than the rest. Overall 100% soy yogurt contained the lowest

percentage of protein among all the yogurt samples.

Table 3.4 Compositional analysis (%) of germinated and non-germinated soy-fortiﬁed

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

yo gurts after manufacturing
Sample Protein Fat Carbohydrate Dry Ash Neutral
Matter Detergent
Fiber
I252 5.98 a 0.98 a 1399 1 0,01‘1 23.92 a 3.02 a 0.92 a
003° 0.06° 014° 0.1 1° 002°
169 5.87 a 1.13 :r 13,62 1 0,04d 23.73 a 3.12 a 0.74 a:
0.1 1° 002° 0.04f 013° 011°
344 5.16i 0.14 a 17.78 1 0,011’ 25.63 a 2.58 a 0.74 a
0.00°d 006°° 004° 005°° 0.1 1°
159 5.08zt 0.46 a 17.49 1 029b 25.21 a 2.20 a 0.54 a
005° 023° 001° 056° 013°
817 6.82 a 0.40 a 1939 ,1 0,108 29.52 a 2.92 a 0.63 :1:
016° 008° 004° 002° 0.01°°
894 4.80 d: 1.32 1 15.72 a: 037° 23.91 i 2.08 i 1.60 :1:
018° 0.02° 005° 016° 0.01°
949 5.56 a 0.00 a 17.75 i 0,03b 27.40 a 4.09 a: 0.00 a
0.1 1°° 000° 001° 008° 0.00d

 

1“Mean values in a column with different letters are signiﬁcantly different (p< 005);

11 = 2 for all samples

1252 = 50% germinated Vinton 81 + 50% non-fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk
344 = 50% germinated DF 222 + 50% non-fat dry milk
159 = 50% non-germinated DF 222 + 50% non-fat dry milk

817 = 50% germinated E05276-T + 50% non-fat dry milk

894 = 100% non-germinated Vinton 81 (all soy yogurt- control)
949 = 100% non-fat dry milk (all dairy yogurt- control)

89

 

3.4.3.2 Eﬂect of soy fortiﬁcation on fat content of yogurt

Since only non-fat dry milk was utilized for the formulation of 100% dairy yogurt,
the fat content was 0%. On the other hand, 100% soy yogurt formulated with non-
germinated Vinton 81 spray dried soy powder and non-germinated/ germinated Vinton 81
fortiﬁed yogurts had the highest fat contents (1 .32%, 1.13%, and 0.94% respectively),
which were statistically signiﬁcant from the rest of the yogurt samples. Yogurts fortiﬁed
with soy powder varieties of germinated E05276-T, germinated and non-germinated DF
222 had lower fat contents than yogurts fortiﬁed with Vinton 81 powders (Table 3.3).
This result is expected Since the yogurts were formulated from non-fat dry milk and low
fat soybean powders. This data suggest that whatever fat is in the yogurt was from the
soy powder utilized.

Although the fatty acid proﬁle of these fats in the present study is not known,
previous studies indicate that the fat contents of soy contain mostly unsaturated fatty
acids (USDA, 1979; Eitenmiller, 1997). The yogurts made from these formulations could
be regarded as non-fat (less than 0.5 g/8erving milk fat) or low fat (not more than
2 g/3erving milk fat) soy fortiﬁed yogurts, which could be beneﬁcial to consumers with
cardiovascular diseases. In the United States of America, yogurts are identiﬁed according
to the standards of identity listed in the U S. Code of Federal Regulations (CFR), in

sections 21 of the Food and Drug Administration (NY A, 2003).

3. 4.3.3 Eﬂect of soy fortiﬁcation on carbohydrate content of yogurt
The carbohydrate contents were obtained from the difference between the dry

matter and the protein, fat and ash contents. Germinated E05276-T fortiﬁed yogurt

90

sample had the highest carbohydrate content that was statistically signiﬁcant ﬁ'om the
rest. All dairy control (100% non-fat dry milk) yogurts’ carbohydrate content was
17.75% and it was similar to the carbohydrate contents of sample 344 (17.78%) and
sample 159 (17.49%). The carbohydrate contents of the yogurt varieties made ﬁ'om
germinated and non-germinated Vinton 81 (samples 252 and 169) were the lowest i.e.

13.99% and 13.62% respectively.

3.4.3.4 Eﬂect of soy fortification on the ash and neutral detergent ﬁber contents of yogurt

The 100% dairy yogurt, sample 949 contained the highest amount of ash (4.09%),
but had 0% neutral detergent ﬁber. Overall, the ash contents of soy fortiﬁed and all soy
yogurt samples were similar. This trend was also seen in the ﬁber contents although the
ﬁber content of 100% soy yogurt was the highest (1 .6%). Sensory evaluation of some
these yogurt samples have suggested likely acceptance of products. With the increase in
public’s interest in! wellness, health and functional foods, the future of soy-fortiﬁed
yogurts is promising.

Dairy yogurt fortiﬁcation with ﬁbers, iron and other selected nutrients in order to
enhance health beneﬁts have been studied (F emandez-Garcia and others 1998; Hekmat
and McMahon 1997). Yogurts fortiﬁed with 2.5% soy protein were found to be similar in
sensory, chemical and microbiological properties to traditional dairy yogurts (Drake and
others, 2000). Our sensory evaluation showed similar results where all the blended
yogurts were similar in overall acceptance with the all-dairy control yogurt. Some
workers in their study showed that consumer attitudes towards acceptability of yo gurts

fortiﬁed with soy powder increased with increased frequency of dairy yogurt

91

consumption as well as increased knowledge of the health claim associated with soy
products (Drake and Gerard, 2003). This observation was made during the sensory
exercise for this study based on verbal comments made by the panelists.

In summary, the results obtained in this objective clearly show that dairy yogurt
could be fortiﬁed up to 50% with whole soy powder. The most desirable yogurt was
manufactured with soybean variety DF 222. Flavor is a major challenge faced by product
developers especially when ingredients containing proteins are incorporated into food
products. More work is needed to increase the acceptability scores of the blended yogurt
samples for example by increasing the moisture content (decrease viscosity) and

ﬂavoring compound without compromising the nutrient composition of the yogurt base.

92

CHAPTER4

PRODUCTION AND CONCENTRATION OF GENISTEIN, DAIDZEIN,
GENISTIN, DAIDZIN AND STACHYOSE IN PREDIGESTEDIGERMINATED

AND NON-GERMINATED SOY POWDER

4.1 ABSTRACT

The concentrations of isoﬂavone isomers genistein, daidzein, genistin and daidzin
were determined in raw, non-germinated and germinated soy powders of Vinton 81, DF
222 and E05276-T soybean varieties. Reverse-phase high-performance liquid
chromatography (HPLC) with ultraviolet (UV) detector was used to determine the
concentration of the isoﬂavones present in the soy powders. Standard curves were made
for each isoﬂavone standard and these curves were utilized to ascertain the concentration
of the isoﬂavones in the samples. The extracted analytes were separated on a C-18
reverse phase column, eluted with acetonitrile/HPLC grade water/Acetic acid (75/25/ 1,
v/v/v), and detected by UV detector at 262nm wavelength. Similarly oligosaccharide,
stachyose contents of the soy powders was analyzed using HPLC.

Nutrient composition analysis Showed that the protein contents of all soy powders
were between, 50.28% to 56.03%. All the isoﬂavones measured were present but genistin
and daidzin were more abundant than genistein and daidzein in all the powders analyzed.
’Mean total isoﬂavones in the soybean varieties ranged ﬁom 290 ug/ g (in raw E05276-T)
to 726 jig/g (in non-germinated DF 222). Soaking and germination increased (p< 0.05)

the isoﬂavone contents and decreased (p< 0.05) the stachyose contents of all soybean

93

varieties, thus suggesting that processing methods could inﬂuence the concentrations of
these compounds. The genistein and daidzein contents of all the raw powders were
similar statistically and ranged from 0.0 ug/ g (genistein in E05276-T‘) and 5.111g/ g
(genistein in Vinton 81); 2.1pg/g (daidzein in Vinton 81) and 3.7 pig/g (daidzein in DF
222). There was a signiﬁcant reduction in stachyose content (53.7% for Vinton 81; 66.9%

for DF 222) after germination.

4.2 INTRODUCTION

Isoﬂavones are phenolic compounds with similar structure to human estrogen
(Tsangalis and others, 2002). Soybeans and non-fennented soy foods contain these
isoﬂavones mostly in the form of biologically inactive conjugates consisting of 83.90%
to 98.37% of the total isoﬂavones (King and Bignell, 2000). These isoﬂavones especially
the aglycone isomers confer health beneﬁts against several aging diseases, cardiovascular
diseases, high cholesterol, osteoporosis, prostrate, colon and breast cancers. Isoﬂavones
can induce biological responses that are capable of mimicking endogenous estrogens by
binding to estrogen receptors (Messina and Hughs 2003). The concentration range of
isoﬂavones is < 50ug/ g to > 3500ug/ g in both soybean seeds and products but the
glucoside forms are higher in concentration in these products. Also, the molecular weight
of the glucoside is twice that of the corresponding aglycones.

The concentrations of the aglycones namely genistein, daidzein and glycitein are
low in soy foods, about 0.2-1 .Smg/ g (Wang and Murphy, 1994). These aglycones are
known to be the most bioactive phytochemicals in soy because of their unique properties.

They are easily absorbed in higher amounts than the other isomers in the gut (Izurni and

94

others, 2000). According to these workers, the gut microﬂora and the enzyme
glucosidases produced by these microorganisms convert the glucosides into
corresponding aglycones. The type and quantity of each isoﬂavone varies according to
the product and processing procedures utilized (Song and others, 1998). Overall genistein
is more abundant in soy foods than daidzein. Fermentation, heat and enzyme treatment
could signiﬁcantly change the isomeric forms of isoﬂavones (total of 12 isomers) (Song
and others 1998). In order to claim health beneﬁts, the suggested amount of isoﬂavone
aglycone required is about 30 to 40mg/day (Malnig and Brown 2007), hence it is
important to provide foods with a considerable amount of aglycones. Germination leads
to endogenous enzyme activation resulting in hydolysis of soy proteins, isoﬂavones and
other boactive molecules (Bau and others 2000).

Several techniques have been employed in isoﬂavone measurement in the past but
most are time consuming and challenging. One of the methods utilized is gas
chromatography with mass spectrometric detection (GC-MS), but the disadvantage of
this method is that the compounds need to be derivatized before injection (Preinerstofer
and Sontag, 2004). As a result, the AOAC international developed a more reliable
method that could be used to measure isoﬂavone levels in foods using reverse phase-high
performance liquid chromatography (RP-HPLC) and ultraviolet (UV) detector
(V erbruggen and others 2002). RP-HPLC is very popularly used for the identiﬁcation of
isoﬂavones in soybeans and soy foods because it does not require any derivatization as
aglycones or glycosides. This method uses mixtures of methanol or acetontrile and

aqueous acids or buffers as mobile phase.

95

The extraction of isoﬂavones from food matrices is generally done with glacial or
common organic solvents such as methanol, acetonitrile, ethanol and acetone in aqueous
medium e.g. distilled water (Murphy and others 2002; Song and others, 1998). Different
temperatures with or without agitation could be used during extraction in order to
enhance efficiency. Isoﬂavone conjugates are very unstable and investigations by
Coward and others (1998), revealed that elevated temperatures enhance isoﬂavone
recovery and de-esterify the malonyl group, where as room temperature extraction
slowed down the conversion of one form to another. This study also showed that
isoﬂavone extraction at reﬁigeration temperature (4 °C) for 2-4 hours gave the highest
amount of malonyl glucoside conjugates and lowest amount of B-glucoside forms.

Soybeans contain indigestible oligosaccharides like rafﬁnose and stachyose, which
are usually associated with ﬂatulence and other stomach discomfort (Rackis and others
1970). Soaking and sprouting of legume seeds can improve starch digestibility and thus
reduce the amount of oligosaccharides through the release of a—galactosidase (Aranda and
others 2001). Stachyose and other indigestible oligosaccharides can reach the colon intact
and are capable of acting like prebiotics, therefore stimulating the growth of probiotics
such as biﬁdobacteria and lactobacilli (Crittenden and Playne 1996). These
oligosaccharides also have physiological effects Similar to dietary ﬁber. Doses of 15 g/day
have been demonstrated to enhance the growth of probiotics while consumption of 40 to
50g/day can cause intestinal discomfort (N yman 2002).

To be able to estimate how much isoﬂvones and stachyose are available to
consumers, it is important to know the concentration levels of these compounds in soy-

based products in order to maximize the health beneﬁts of soybean consumption. The aim

96

of this study therefore is to determine the concentration of genistein, daidzein, genistin,
daidzin and Stachyose in germinated, non— germinated and raw (untreated) soy powders

from Vinton 81, DF 222 and E05276-T soybean varieties as potential food ingredients.

4.3 MATERIALS AND METHODS
4.3.1 Materials
4.3.]. I Chemicals and solutions

Genistein, daidzein, geinstin, daidzin standards, glacial acetic acid (9999+ %
purity), and dimethyl sulfoxide (99.9%% purity) were purChased from Sigma-Aldrich (St.
Louis, Missouri, USA). Methanol (HPLC grade) was bought from J .T. Baker
(Mallinckrodt Baker Inc., Phillipsburg, NJ, USA) and acetonitrile (HPLC grade), was
from EM Science (EM Industries, Inc., Gibbstown, NJ, USA). HPLC water used was
obtained from nanopure inﬁnity ultrapure water system (Barnstead/Thermolyne
Corporation, Dubuque, IA, USA).

Several stock solutions of genistein, daidzein, genistin and daidzin standards were
prepared. Stock glucoside standards were prepared by weighing 20 mg each of genistin
and daidzin reference standard into a 10 ml volumetric ﬂask. Similarly, stock aglycones
standards were prepared by weighing 5 mg each of genistein and daidzein reference
standard into 10 ml volumetric ﬂask. Eight milliliters of dimethyl sulfoxide was added to
each ﬂask and sonicated until completely dissolved and each mix was then diluted to
volume with dimethyl sulfoxide and mixed. Mixed standard dilutions were created using
methanol/water (80/20) and the stock standards were diluted to create a ﬁve-point curve.

The following dilutions of each glucoside and aglycone stock with methanol/water were

97

prepared: 2/ 10, 1/ 10, 1/25, 1/50, and 1/ 100 (concentration varied from 5.05 to 500ug/g).
All the standards were stored at refrigeration temperature and protected from light for up
to 60 days.

The mobile phases consisted of two solvents. Solvents A and B were prepared
mixing water/acetonitrile/acetic acid in the following ratios respectively 75:25:] and
25:75: 1. The mixtures were degassed by soniﬁcation for 5 minutes prior to use in the
HPLC analysis.

4. 3 . 1 .2 Instrumentation

The HPLC system consisted of the standard for isocratic and multiple pump
gradient systems containing, two HPLC pumps (Waters 1525 Binary HPLC Pump), an
HPLC autosampler (Waters 717 plus Autosampler), HPLC dual wavelength 0.)
absorbance detector (Waters 2487), a hydrosphere C18 column 150 x 4.6 mm, particle
size 5 pm and a hydrosphere C18 guard column 4 x 20 mm, particle size Sum (Waters
Corporation, Milford, MA, USA). A Dell compatible computer installed with Waters

Breeze chromatography software (Version 3.30) was used to control the HPLC system.

4.3.2 Methods
4. 3.2. 1 Calibration curves and calculation of standard solutions

Five hundred microliters of each dilution was added into 1 ml clear glass shell vial
with polyethylene snap cap (Waters Corporation, Milford, MA, USA). Ten microliters of
each standard solution (ﬁve dilutions for each standard) were injected into the
chromatographic system. The compounds were separated using the gradient system on a

C 18 column attached to a C18 guard column at 37 °C using a ﬂow rate of 1.0 ml/min. A

98

UV detector was used to detect the compounds at a Single wavelength of 262 nm and set
at 0.08 AUFS (absorbance units). The injection volume was 10 11] and the following
gradient was used: 0-15 min, 100% solvent A and 0% solvent B; 15-25min, 0% solvent A
and 100% solvent B; 25-27min 100% solvent A and 0% solvent B, and run cycle was 40
min.

The chromato grams were recorded using the data analysis Breeze software
(Waters Corporation, Milford, MA, USA). The peak areas were used to create a plot of
standard peak areas versus standard concentrations (standard curve) for each isoﬂavone
standard (6. g. Figures 4.1 and 4.2). Replicate standard curves were obtained and these
curves were used to calculate the concentrations of the isoﬂavone isomers that were
extracted from the whole soy powders. The retention time and UV absorption patterns of
pure isoﬂavones were used to identify isoﬂavones in the soy powder samples.

Figure 4.1 Standard curve for pure genistein standard

4000000

 

   

3500000 - y = 27645x + 150426
R2 = 0.9975

 

3000000 .

2500000 —
N
2 2000000 ~
<

1500000 -

1000000 -

500000 —

 

 

0 I l T I l
0 20 40 60 80 100 120 140
Conc. (uglg)

 

99

 

 

Figure 4.2 HPLC chromato gram for pure genistein standard at different concentrations

(average retention time = 19.956 minutes.)

 

0.5 1:

 

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100

Figure 4.2 continued.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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0.06“ at
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Mnutes

101

 

 

4.3.2.2 Proximate analysis of soy powders

Compositional analyses of the germinated and non-germinated soy powders were
done using standard or slightly modiﬁed standard methods. These analyses were carried
out in the Animal Science Department laboratories. Crude fat, protein, carbohydrate (by
subtraction), ash, neutral detergent ﬁber and dry matter were determined. Ether extraction
method was used for the crude fat analysis, neutral-detergent ﬁber was analyzed (Goering
and Van Soest, 1970; Robertson and Van Soest, 1977; Chemey and others, 1989). The
hach total nitrogen (modiﬁed kjeldahl digestion) method was used for the protein analysis
(Hach and others, 1987). Ash content was determined using the AOAC (2005) method

945.46.

4.3.2.3 Isoﬂavone extraction

Spray-dried germinated and non- germinated soy powders and raw ground
soybeans were analyzed. The soybean varieties utilized were Vinton 81, DF 222 and
E05276-T. A method adopted by Bennink and Barret (2004) was used for the extraction.
Two grams of each soy powder was weighed into 50 ml centrifuge tube and 35 ml of an
extraction solvent made up of 50% methanol: 49% ultrapure water: 1% acetic acid was
added to the powder and mixed with spatula and sonicated for 30 min. The mixture was
centrifuged at 4,000 rpm for 10 min and the supernatant was poured into a centrifuge
bottle (Figure 4.3). Further extractions were repeated using the following extraction
solvents: 70% methanol: 29% ultrapure water: 1% acetic acid; 90% methanol: 9%
ultrapure water: 1% acetic acid; 100% methanol. All the combined supematants were

kept in the centrifuge bottles overnight. Aﬁer overnight storage the bottles were

102

centrifuged at 5,000 rpm for 15 min. The corresponding supematants were poured into
500 ml round bottom ﬂasks.

The ﬁltrate for each extracted sample was dried almost completely on a rotary
evaporator (Biichi; Brinkmann, Westbury, NY) at approximately 45 °C. The residue was
dissolved in 15 ml 80% methanol and sonicated. The dissolved samples were ﬁltered
through 0.45 pm Supor membrane disc ﬁlters (Waters Corporation, Milford, MA, USA).

All the ﬁltered samples were stored in the ﬁeezer (-20 °C) prior to HPLC analysis.

4.3.2.4 Reverse-phase high performance liquid chromatography (HPLC) of isoﬂavones
Waters plus C-l8 SEP-PAK ﬁlter cartridges (pore size 0.22 pm) (Waters
Corporation, Milford, MA, USA) were used for HPLC analysis. Each ﬁlter cartridge was
conditioned with 5 m1 of 80% methanol and 5 ml of HPLC grade water (nanopure water)
before sample application. A portion (1 m1) of each evaporated and re-dissolved sample
was micro ﬁltered into 1 ml clear glass shell vial with polyethylene snap cap (Waters
Corporation, Milford, MA, USA) and injected into HPLC system. The same running

condition with the standards was also applied to the samples in the HPLC system.

4.3.2.5 Reverse-phase high performance liquid chromatography (HPLC) of stachyose
Stachyose contents of germinated and non-germinated soy powders were

evaluated by ABC Research Corporation (Gainsville, FL, USA). Extraction method by

Omogbai and others (2005) were utilized before HPLC analysis. It involved the use of 2-

propanol for extraction. Supernatant was ﬁltered after centrifuging before HPLC analysis.

103

Figure 4.3 Flow diagram for isoﬂavone extraction from soybean powders

Weigh 2 g soy powder
Add 35 ml of 500/1 methanol solvent
Sonicate for 30 min at ambient temp.
Centrifuge at 4,000 rpm for 10 min. to extract isoﬂavone
Pour supernatant into centrifuge bottle and label
Add 35 m1 of 70% methanol solvent
Repeat steps 3, 4 and 5

Add 35 ml of 90% methanol solvent

1

Repeat steis 3,4 and 5

Add 35 m1 100% methanol solvent
Repeat steps 3, 4 and 5
Add 30 ml of 100% methanol to all combined supematants in bottles
Store in the reﬁigerator (4 °C) overnight prior to evaporation
Centrifuge at 5000 rpm for 15 min., dry supernatant in rotary evaporator

l

Dissolve residue in 15 ml 80% methanol, ﬁlter through 0.45pm ﬁlter prior to HPLC

104

4.3.3 Statistical analysis

Replicate data for nutrient composition and stachyose, and triplicate data for
isoﬂavone analysis were used for all the analysis. Statistical analysis including linear
regression, average and standard deviation (SD) was performed using the statistical
function of Microsoft Excel and Sigma plot 9.0(Jandel Scientiﬁc, San Rafael, CA).
Analysis of variance was done using Tukey’s test for multiple comparisons of the means.

Sigma Stat 3.1(Jande1 Scientiﬁc, San Rafael, CA) was utilized for this analysis.

4.4 RESULTS AND DISCUSSION
4.4.1 Effect of germination on compositional analysis of soybean powder

The proximate analysis data is shown in Table 4.1. Each data represents means of
two replicates. The average protein content of whole soybeans is about 40% with about
20% fat, 35% carbohydrate and 5.0% ash (Min and others 2005). Our results indicated
that the protein contents of all the soybean varieties utilized were between 50.28 to
56.03% well above the average. The soybean seed varieties used for this study contained
35.5% protein and 19.8% fat (DE 222), 39.1% protein and 17.6% fat (Vinton 81) while
the E05276-T variety contained 36.7% protein and 16% fat in the raw beans. According
to several workers such as Khalil and others (2006), Bau and others (2000), Wang and
others (2003), the nutrient contents and protein digestibility can be improved and anti-

nutritional factors reduced when soybeans were soaked, germinated and dehulled.

105

Table 4.1 Nutrient composition (%) of germinated and non-germinated soy powders

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Protein Fat Carbohydrate 11:1?tter Ash 13:32:11:
Fiber
‘Gv 31 56.03 i 20.85 a: 18.80 a: 0.30”I 92.41 :t 4.32 :t 4.54 :1:
0.35a 0.01” 0.01” 0.63” 0.058
NGV 81 50.76 :t 18.39 :t 26.06 :1: 0.65”” 92.17 a 4.79 :1: 4.39 :1:
0.49” 0.01” 0.28” 0.16”” 0.04a
GDF 222 51.00 a 22.40 :1: 22.20 at 036° 92.25 :1: 4.40 3: 4.55 :1:
0.68” 0.44d 0.15” 0.14” 0.04a
NGDF 50.28 :1 17.62 :t 27.8121: 0.88” 93.22 :1: 5.09 :I: 4.39 :1:
222 0.95” 0.05” 0.15a 0.02a 0.09a
GET 54.97 :I: 15.81 i 24.20 3: 0.26”c 92.60 :1: 5.02 a: 3.84 :t
0.03a 0.13a 0.13”” 0.16” 0.02”

 

“1 Means in the same column with small letter superscripts are signiﬁcantly different
(p<0.05); n = 2 for all samples
lGV 81 = Germinated Vinton 81

NGV 81 = Non-germinated Vinton 81

GDF 222 = Germinated DF 222

NGDF 222 = Non-germinated DF 222

GET = Germinated E05276-T

According to Khalil and others (2006), their study suggested that enzyme

hydrolysis during and after germenation of raw soybeans resulted in a remarkable

increase in the protein content. Our data clearly showed that soaking and germination

greatly improved the nutrient contents especially the protein contents. Ahmad and Pathak

106

 

(2000) also reported that no signiﬁcant changes were observed in protein, total sugars,
reducing sugars or ash contents of germinated and non-germinated soybean ﬂours.

Overall the germinated soybeans in this research had higher protein contents than
the non-germinated counterparts although germinated Vinton 81 (56.03 %) and
germinated E05276-T (54.97%) were signiﬁcantly higher statistically (p < 0.05), than the
rest (Table 4.1). There was no signiﬁcant difference in protein content between
germinated and non-germinated DF 222 powders. Soybean proteins have played a
signiﬁcant role in human nutrition for a long time in both developing and developed
countries. Soaking and germination process, induced increase in hydrolytic enzymes
(increased water activity), which facilitated the metabolism of nitrogenous compounds
from carbohydrate reserves, probably leading to increase in crude protein contents (Khalil
and others 2006). On the other hand, the carbohydrate contents of the non-germinated
soybeans were signiﬁcantly higher than the germinated counterparts. Non-germinated
Vinton 81 and DF 222 contained 26.06% and 27.81% carbohydrate respectively while
their germinated varieties had 18.80% and 22.20% carbohydrate respectively. This
observation could be as a result of lower a-galactosidase activity in the non-germinated
soybeans hence the carbohydrate contents remained more intact than the germinated
soybeans (Bau and others 2000). Such soybeans as previously discussed are expected to
have ﬂatulence-causing properties.

The total fat contents were generally low in all varieties although it is expected
that the lipoxygenase activities should be lower in germinated than in non-germinated
soybeans therefore improve odor and ﬂavor of such beans. The fat content of germinated

E05276-T soy powder was signiﬁcantly lower than the rest of the powders. Non-

107

germinated Vinton 81 and DF 222 had signiﬁcantly lower fat contents (18.39% and
17.62% respectively) than their germinated varieties (20.85% and 22.40% respectively).
The ash contents of the non-germinated soy powders were generally higher than the
germinated powders. Ash contents varied between 4.32% (for germinated Vinton 81) to
5.09% (for non-germinated DF 222). Similarly there was no signiﬁcant difference
between the dietary ﬁber contents of germinated and non-germinated Vinton 81 and DF
222 (Table 4.1). The dry matter, which is an indication of the moisture contents of the
powders, varied from 92.17% (for non-germinated Vinton 81) to 93.22% (for non-

germinated DF 222).

4.4.2 Effect of germination on total isoﬂavone content

The average total isoﬂavone (4 isomers, on dry matter basis) contents, namely
genistin, genistein, daidzin and daidzein of raw, non-germinated (soaked) and germinated
soybean varieties are shown in Table 4.2.

Table 4.2 Total isoﬂavone contents (on dry matter basis) in raw and spray dried
germinated and non- germinated soybean powder ( pg/ g)

 

Soybean varieties

 

 

Seed treatment Vinton 81 DF 222 E05276-T

Raw 378.74 :I: 1.32” 375.33 :1: 19.85° 290.13 a 28.20”
Non-germinated (soaked) 509.41 :1: 19.41a 726.16 a 51.808 N/A*
Germinated 419.84 :1: 2.55” 414.77 :1: 28.83” 611.87 i 21.30”

 

3") Values in a column with different letters are signiﬁcantly different (p< 0.05)

* Not Available

108

The total isoﬂavone contents of the isomers measured in the raw beans from
soybean varieties Vinton 81, DF 222 and E05276-T were 378.74, 375.33 and 290.13
ug/ g, respectively. The total isoﬂavone contents in Vinton 81 and DF 222 varieties
increased after soaking. During the course of this study, the limited availability of
E05276-T variety did not allow us to process the non-germinated type hence there was no
data for this treatment. Among the germinated powders, the total isoﬂavone content of
E05276-T was the highest (611.87 pg/ g). There was signiﬁcant difference between the
total isoﬂavone contents of the raw and germinated soy powders of Vinton 81 and DF
222 and E05276-T varieties.

According to Zhu and others (2005), the isoﬂavone contents of soybeans are
affected by soybean variety, environmental changes like temperature, and amount of
sunshine and moisture level. The increase of isoﬂavone contents within the same soybean
variety could be attributed to induced metabolic pathways of the precursors of
isoﬂavonoids commonly found in legumes or oil seeds.The formation of B-glucoside is
increased as a result of de-esteriﬁcation of malonyl- and acetylglycoside. As observed in
Table 4.2, the increase after soaking and decrease after germination of isoﬂavone
contents could be due to the conversion of different ﬂavonoids to isoﬂavones and vice
versa or conversion of an isoﬂavone isomer to another isomer as reported by Terrence

(1991).

4.4.3 Effects of germination on Genistein and Genistin contents

Genistein and its B-glucoside conjugate, genistin contents in variously treated soybean

powder varieties are shown in Table 4.3. Total genistein and genistin increased

109

signiﬁcantly during soybean soaking and germination. The maximum amount was
obtained at soaking (354.2 pg/g) for variety DE 222, and 285.2.ug/g for variety Vinton
81. Data was not obtained for the soaked version of E05276-T variety because the
powder was not available but its germinated counterpart had a total content of 316.2
ug/g, which was signiﬁcantly higher than the raw powder (137.6 ug/ g).

Table 4.3 Total Genistein and Genistein contents in raw and spray dried germinated and

non-germinated soybean powder (pg/ g)

 

 

 

 

 

 

 

 

 

 

Soybean variety Genistein Genistin Total

‘GV 81 20.091 i 1.38”” 220.740 :1: 4.44” 240.831 i 6.99”
NGV 81 59.154 i 7.77” 226.055 .4.- 4.25” 285.209 3. 20.76””
RV 81 5.069 i 2.63” 172.075 :1 1.37” 177.144 :t 3.86”
GDF 222 66.123 1 8.12” 177.551 a 6.72” 243.674 :1: 25.21”
NGDF 222 55.528 :1: 9.49” 298.65 a: 10.27” 354.174 :1: 34.19”
RDF 222 3.025 a 2.26” 165.250 3: 3.10”” 168.275 :1: 10.35”
GET 42.195 3: 12.79”” 274.017 i 6.99” 316.212 i 23.21””
RET 0.000 :1 0.00” 137.608 :1: 6.75” 137.608 :1: 11.693”

 

 

 

 

 

8'1) Values in a column with different letters are signiﬁcantly different (p< 0.05); n = 3

1GV 81 = Germinated Vinton 81
NGV 81 = Non-germinated Vinton 81
RV 81 = Raw Vinton 81

GDF 222 = Germinated DE 222
NGDF 222 = Non-germinated DF 222
RDF 222 = Raw DF 222

GET = Germinated E05276-T

RET = Raw E05276-T

110

There was a highly signiﬁcant difference in genistein content between the raw and
soaked or germinated varieties. Raw seeds from E05276-T seem to have no detectable
amount of genistein (0.00 ug/ g). The differences especially in the genistein content
among raw, non-germinated (soaked only), and germinated soy powders could be as a
result of physiological changes that occurred during soaking and germination. Soaking
and germination to a limited extent induce the hydrolysis of glucosides, which leads to an
increase in genistein content (Zhu and others, 2005). The decrease in genistein content in
germinated Vinton 81 when compared to non-germinated Vinton 81 could be as a result

of the conversion of its genistein to other isoﬂavones.

4.4.4 Effects of germination on Daidzein and Daidzin contents

The effect of soybean germination on daidzein and daidzin is shown in table 4.4.
All the raw soybean varieties had similar daidzein content i.e. Vinton 81 (2.13ug/g), DF
222 (3.65 ug/ g) and E05276-T (2.49 pig/g). Overall, a small quantity of daidzein
(aglycone) was present in the soy powders in comparison with its glucoside component
i.e. daidzin. The maximum amounts of daidzein (27.95 and 27.37 ug/ g) were observed
after soaking and germination in DF 222, which was not signiﬁcantly different (p < 0.05)
from non-germinated (soaked only) Vinton 81 (21.52 ug/g), and germinated E05276-T
(18. 99 pg/ g). Germinated Vinton 81 had signiﬁcantly lower daidzein content (5.65 ug/ g)
than the non-germinated counterpart. This observation was also similar to the result
obtained in the isoﬂavone, genistein content in germinated and non- germinated Vinton

81.

111

Table 4.4 Total Daidzein and Daidzin contents in raw and spray dried germinated and
non-germinated soybean powder (ug/ g)

 

 

 

 

 

 

 

 

 

 

 

 

 

Soybean variety Daidzein Daidzin Total

IGv 81 5.650 1 1.07” 173.355 1 3.53”” 179.006 1 4.08”
NGV 81 21.516 1 3.27” 232.685 1 31.82”” 224.202 1 0.81”
RV 81 2.129 1 1.07” 199.468 1 1.87”” 201.597 1 2.57”
GDF 222 27.370 1 4.44” 143.733 1 0.81” 171.103 1 5.07”
NGDF 222 27.955 1 3.61” 344.044 1 10.64” 371.999 1 10.18”
RDF 222 3.651 1 0.05” 203.400 1 5.13” 207.051 1 5.53”
GET 18.994 1 2.249” 276.666 1 2.66” 295.660 1 3.31”
RET 2.494 1 0.44” 150.031 1 9.70” 152.525 1 9.74”

 

3") Mean values in a column with different letters are signiﬁcantly different (p< 0.05);

=3

1GV 81 = Germinated Vinton 81

NGV 81 = Non-germinated Vinton 81
RV 81 = Raw Vinton 81

GDF 222 = Germinated DF 222
NGDF 222 = Non-germinated DF 222
RDF 222 = Raw DF 222

GET = Germinated E05276-T

RET = Raw E05276-T

112

 

The percent increase of daidzein contents in non-germinated i.e. soaked and spray-
dried soy powders from varieties Vinton 81 and DF 222 were 910.6% and 665.7%
respectively compared to raw powders. Similarly, the percent increase of daidzein
content in germinated powders from Vinton 81, DF 222 and E05276-T varieties were
165.4%, 649.7% and 661.6% respectively compared to raw powders. However there was
a remarkable decrease in daidzein content between non-germinated and germinated soy
powders for Vinton 81. Germinated DF 222 and germinated E05276-T powders had
signiﬁcantly higher daidzein content (27.96 and 18.99 ug/ g respectively) than germinated
Vinton 81 powder (5.65 pg/g), which was not signiﬁcantly different (p < 0.05) from raw
powders from all the soybean varieties. The total amount of daidzein and daidzin were
signiﬁcantly higher in non-germinated powders (p < 0.05) in DF 222 and Vinton 81.
Non-germinated powder from E05276-T was not available hence no data was obtained.
The maximum amount of daidzein and daidzin was observed in non-germinated DF 222
variety (372 ug/g).

The retention times of four soy isoﬂavone isomers measured are shown in Figures
4.4, 4.5 and 4.6. The retention times for daidzin, genistin, daidzein and genistein were
approximately 13.7, 14.9, 17.6 and 19.8 minutes respectively. Overall, the B-glucosides
isomers genistin and daidzin were more abundant than the aglycone isomers genistein
and daidzein in all the soy powders analyzed (Figure 4.7). This could be to due the fact
that the aglycone forms are easily converted during processing and storage into other
isoﬂavone isomers that were not measured, but are equally beneﬁcial to the health of the
consumer (Coward and others, 1998). Generally, the total amount of isoﬂavones in each

powder was high.

113

Figure 4.4 Representative HPLC chromatogram of isoﬂavones in germinated (GV 81),
non-germinated (NGV 81) and raw (RV 81) Vinton 81 soybean varieties (a, daidzin; b,

genistin; c, daidzein; d, genistein).

 

 

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114

Figure 4.5 Representative HPLC chromatogram of isoﬂavones in germinated (GDF 222),
non-germinated (NGDF 222) and raw (RDF 222) DF 222 soybean varieties (a, daidzin;

b, genistin; c, daidzein; d, genistein).

 

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115

Figure 4.6 Representative HPLC chromatogram of isoﬂavones in germinated (GET), and
raw (RET) E05276-T soybean varieties (a, daidzin; b, genistin; c, daidzein; d, genistein).

 

 

 

 

 

 

 

 

 

 

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116

Figure 4.7 Concentrations of soy isoﬂavone isomers in germinated, non-germinated and

raw soy powders.

 

 

 

 

 

 

 
   

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

400
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Genistein Daidzein Genistin Daidzin

lsoflavone isomers

GV 81 = Germinated Vinton 81

NGV 81 = Non-germinated Vinton 81
RV 81 = Raw Vinton 81

GDF 222 = Germinated DF 222
NGDF 222 = Non-germinated DF 222
RDF 222 = Raw DF 222

GET = Germinated E05276-T

RET = Raw E05276-T

117

4.4.5 Effects of germination on Stachyose contents

Stachyose contents of germinated and non-germinated soy powders are shown in
Table 4.5. All the germinated soy powder varieties i.e. Vinton 81, DF 222 and E05276-T
had signiﬁcantly lower (p <0.05) amounts of stachyose (19.15 mg/g, 15.45mg/ g and
13.9mg/ g respectively). On the other hand, non-germinated Vinton 81 and DF 222
contained 41.35 mg/ g and 46.65 mg g respectively.

Table 4.5 Stachyose contents in spray dried germinated and non-germinated soybean

 

 

 

Powder (mg/g)

Soybean varieties
Seed treatment Vinton 81 DF 222 E05276-T
Non-germinated (soaked) 41.35 :1: 0.92b 46.65 :1: 0.788 N/A*
Germinated 19.15 :1: 0.35c 15.45 :t 0.35"d 13.90 i 2.26d

 

“1 Values with different superscript letters are signiﬁcantly different (p< 0.05); n = 2

* Not Available
Mature raw soybeans contain about 1.4 to 4.1% stachyose depending on the

variety. Stachyose is composed of one sucrose molecule connected to two molecules of
galactose. In this study the value of stachyose contents in the germinated powders ranged
from 1.39% (in E05276-T) to 1.91% (in Vinton 81). Meanwhile, the stachyose contents
in non-germinated powders varied from 4.14% (in Vinton 81) to 4.67% (in DF 222),
which were signiﬁcantly different from the germinated powders. These data clearly
showed that germination process greatly reduced the contents of stachyose by 53.7% in
Vinton 81 and 66.9% in DF 222 varieties. This phenomenon could be attributed to the

increase in enzyme a-galactosidase during germination (V iana and others, 2005), which

118

may have resulted in the conversion of the tetrasaccharide to monosaccharides and
disaccharides.

The purpose of this study was to evaluate the effects of germination on isoﬂavone
and stachyose contents and the potential importance on the inclusion of such powders in
food preparations to inﬂuence optimal health beneﬁts. In summary, soaked (non-
gerrninated) and germinated soybeans induced substantial increase in the contents of
isoﬂavones particularly the aglycones. Non—germinated and germinated DF 222 soybean
variety gave the highest aglycone contents. Soaking caused a substantial increase in
daidzin, genistin, genistein and daidzein. In most cases germination of soaked seeds
decreased the aglycone content suggesting that controlled germination (e. g. germination
time, temperature and sprout length) is needed to enhance the aglycone content of
soybean. Also, germination reduced the stachyose contents of the powders signiﬁcantly
compared to non-germination process (soaking only). This suggests that ﬂatulence factor
or stomach discomfort due to stachyose would be reduced in such powders and at the
same time the powders will contain necessary amounts to act as prebiotics or dietary
ﬁbers.

The above results clearly suggest that soybean seed soaking and/or germination
are necessary in order to increases the more bioactive (aglycones) components of soy
food products before consumption. This will lead to increased absorption in the gut of the

consumer hence increase health beneﬁts.

119

CHAPTER 5

EFFECT OF PROCESSING AND REFRIGERATED STORAGE ON
ISOFLAVONE AND STACHYOSE CONTENTS OF YOGURT FORTIFIED
WITH N ON-GERMINATED AND GERMINATED/PREDIGESTED WHOLE

SOY POWDER

5.1 ABSTRACT

The proﬁles of genistein, daidzein, genistin, daidzin and stachyose were
determined in ﬁeeze-dried yo gurts fortiﬁed with germinated or non-germinated spray
dried whole soy powders. The isoﬂavones were evaluated at one week of manufacturing
and also at the end of six weeks of storage at 4 °C while stachyose was evaluated at one
week of manufacturing. The soybean varieties utilized for yogurt making were Vinton 81 ,
DF 222 and E05276-T and reverse-phase high-performance (HLPC) was used for
analysis. The total of four isoﬂavone contents increased aﬁer 6 weeks of storage
compared to the 1St week. Daidzin and genistin (Ii-glucosides) contents contributed most
to the increment during storage. The genistein and daidzein contents of the whole soy-
fortiﬁed yogurts remained signiﬁcantly the same throughout the shelf life period (6
weeks). The isoﬂavones retained in the soy and soy- fortiﬁed yogurts were signiﬁcantly
(p < 0.05) high (e.g. 94% i.e. 478.7 ug/g retained in 100% soy yogurt) at the end of 6‘“
week storage compared to the isoﬂavone contents of the corresponding soy powder

(509.4 ug/ g for non-germinated Vinton 81 powder) that was utilized in the yogurt base.

120

All the yogurt samples containing germinated soy powders had lower stachyose
contents than non-germinated soy-fortiﬁed yogurts (2.82 to 4.41 mg/ g of stachyose). The
stachyose amounts in yogurts fortiﬁed with non-germinated soy powders varied from
8.45 to 17.25 mg/g). The sample with the highest concentration of 17.25 mg/g was the
100% soy yogurt. Hence effect of soybean germination and subsequent fermentation by

lactic acid bacteria during yogurt making was shown in stachyose content.

5.2 INTRODUCTION

Dairy yogurt fortiﬁcation with ﬁbers and minerals so as to provide additional
health beneﬁts has been studied (Fernandez-Garcia and McGregor, 1997; Hekmat and
McMahon, 1997). Yogurt has been considered as a good vehicle to provide combined
beneﬁts of soy protein and dairy ingredients (Schmidt and others 1980). According to the
Federal Register (1999), dairy yogm'ts that contain 5% added soy protein concentrate
meet the FDA requirement for health claim (6.25 g soy protein per serving), while dairy
yogurts that contain only 2.5% soy protein are regarded as a “good source” claim of soy
protein. Previous work showed that dairy yogurts fortiﬁed with 5.0% soy protein was
found to be darker, chalky and less sweet, while yogurts fortiﬁed with 1 or 2.5% soy
protein were most similar to control yogurt (Drake and others, 2000).

There has been considerable interest in soybean isoﬂavones aside ﬁom soy
proteins, and their potential health beneﬁts. Initially, isoﬂavone compositions in soy
foods were thought to be dependent on whether the food was fermented or not. As such it
was assumed that fermented soy foods contained the unconjugated isoﬂavone aglycones

(daidzein, genistein and glycitein) only, while the unfermented foods contained the

121

[El-glucoside conjugates (daidzin, genistin and glycitin) (Coward and others, 1998).
Reverse-phase high performance liquid chromatography (HPLC), has shown that most
soy foods contain mixtures of isoﬂavone isomers. Research has shown that some of these
isomers are altered during food processing and extraction methods.

Major biologically active soy isoﬂavones associated with soy foods consumption
are daidzein, genistein and glycitein as aglycones. Several authors have studied the
biological activity of isoﬂavones and suggested many beneﬁcial roles in the diet
(Setchell, 2002; Song and others, 1998), but there are other studies indicating undesirable
effects of isoﬂavones such as cognitive function, reproductive abilities and breast cancer
risk especially when consumed in high dosages (Sirtori, 2001). Some European countries
like Italy have advised consumers to maintain a daily intake of isoﬂavones, consumed as
dietary supplement lower than 80mg/day (Morandi and others 2005). It has been reported
that 30% to 50% of people in Western countries have the capacity to convert isoﬂavone
glycosides to aglycones and then to equol in the intestinal tract (Frankenfeld and others,
2005; Setchell and others 2002).

Considerable quantities of oligosaccharides present in soy-based foods limit their
biological value and acceptability due to the ﬂatulence-causing factor. At the same time,
these oligosaccharides promote the grth of probiotics in the gut, which promote gut
health. A reduction in oligosaccharide level in soy-based foods will be highly desirable.
Several methods used to lower oligosaccharide contents include hulling, soaking,
cooking, gamma irradiation, germination and microbial or plant a-galactosidase

treatments (Viana and others, 2005).

122

Past studies have shown that soybean soaking and/or germination increased the
aglycone forms of the isoﬂavones. Several workers have also reported the bioconversion
of isoﬂavone glycosides to aglycones in soymilk or fortiﬁed soymilk by probiotics such
as biﬁdobacteria and lactobacilli (Tsangalis and others, 2002; Otieno and others, 2005;
Chien and others, 2006; Shah, 2006; Pham and Shah, 2007; Pham and Shah, 2008). It is
crucial to know exactly what forms and proportion of isoﬂavones the individuals
consume, since it is proposed that aglycones are absorbed more readily than the B-
glucosides. Consequently, dairy/soy-based foods would attract a broader approval if
reasonable amounts of aglycones and oligosaccharides such as stachyose were present. .
The objective of this study was to analyze the yogurt samples and evaluate the outcome
of the bioactive compounds (genistein, daidzein, genistin and daidzin), in the samples

after processing and storage at refrigerated temperature.

5.3 MATERIALS AND METHODS
5.3.1 Yogurt samples

The yogurt samples utilized for this study were freeze-dried in the department of
Animal Science Laboratories, Michigan State University. Brieﬂy, yogurts were prepared
by blending non-fat dry milk with or without soy powders, stabilizer and sugar. The
yogurt mixtures were homogenized and inoculated with Streptococcus salivarius subsp.
thermophilus, Lactobacillus delbreuckii subsp. bulgaricus, Lactobacillus acidophilus
NCF M and incubated at 42 °C until pH of 4.4-4.6. The same yogurt samples were utilized
during the sensory evaluation study and randomly coded as 159 (50% non-germinated DF

222 + 50% non-fat dry milk), 344 (50% germinated DF 222 + 50% non-fat dry milk),

123

169 (50% non-germinated Vinton 81 + 50% non-fat dry milk), 252 (50% germinated
Vinton 81 + 50% non-fat dry milk), 817 (50% germinated E05276-T + 50% non-fat dry
milk), 894 (100% non-germinated Vinton 81- all soy control) and 949 (100% non-fat dry
milk- all dairy control). All the samples were placed in ﬁeezable cups covered with
cheesecloths and labeled appropriately. The cups were placed in a chamber of a
laboratory freeze drier (Sorvall RC 6 Plus, Thermo Electron Corporation, Asheville NC,
USA) to dry at —50 °C and 10 microns Hg pressure until a constant weight was reached.
Total drying was estimated to be 18—26 hours for each load.

5.3.2 Instrumentation and solutions

Reverse-phase high performance liquid chromatography (HPLC) instrumentation
system and conditions utilized in the analysis of isoﬂavone concentration in soybean
powders were also utilized for this study. A gradient system consisting of two HPLC
pumps (Waters 1525 Binary HPLC Pump), HPLC autosampler (Waters 717 plus
Autosampler), HPLC dual wavelength absorbance detector (Waters 2487), a hydrosphere
C18 column 150 x 4.6 mm, particle size 5 pm and a hydrosphere C18 guard column 4 x
20 mm, particle size 5 pm (Waters Corporation, Milford, MA, USA).

Standard curves for each isoﬂavone isomer i.e. genistein, daidzein, genistin and
daidzin standards previously utilized for soy powder isoﬂavone analysis was used for this
study (Figures 4.1 and 4.2). HPLC grade solutions of methanol (J .T. Baker, Mallinckrodt
Baker Inc., Phillipsburg, NJ, USA), acetonitrile (EM Science, EM Industries, Inc.,
Gibbstown, NJ, USA) and nanopure water (Barnstead/Thermolyne Corporation,
Dubuque, IA, USA) were utilized for both extraction and HPLC process. Isoﬂavone

standards were procured from Sigma-Aldrich (St. Louis, Missouri, USA).

124

5.3.3 Extraction and Evaporation

Freeze dried yogurt samples were ground using porcelain mortar and a pestle prior
to mixing. The extraction procedure that was used during isoﬂavone extraction ﬁ'om soy
powders was utilized (Figure 4.3). Here, two grams of ﬁnely ground yogurt samples were
mixed with 35ml of 50% methanol in water and mixed for 30 minutes before centrifuging
at 4,000 rpm for 10 minutes. The supernatant was collected after which subsequent
extractions were carried out with 70% methanol, 90% methanol and 100% methanol
(Figure 4.3).

All the supematants for each sample were combined in a centrifuge bottle and kept
overnight in the reﬁigerator. At the end of overnight storage, the mixtures were further
centrifuged at 5,000 rpm for 15 minutes and the supematants transferred into 500 m1
round bottom ﬂasks. These samples were evaporated in the rotary evaporator (Biichi,
Bimkmann, Westbury, NY) at 45 °C and each residue was dissolved in 15 ml 80%
methanol and mixed thoroughly. Each mixture was ﬁltered through 0.45 um Supor
membrane disc ﬁlters (Waters Corporation, Milford, MA, USA), and all the ﬁltrates were

stored in the freezer at —20 °C pending HPLC analysis.

5.3.4 HPLC Analysis

A linear reversed-phase HPLC gradient used consisted of solvent A, 25%
acetonitrile in water with 1% acetic acid, and solvent B made up of 75% acetonitrile in
water with 1% acetic acid. Same standard curves initially developed for soy powders
(Figures 4.1 and 4.2) for calculation of genistein, daidzein, genistin and daidzin were

utilized for the yogurt sample extracts. Prior to each run, the system was equilibrated for

125

40 minutes with solvent A. The ﬂow rate was 1.0 ml/min. UV spectra (262 nm) were
recorded and area responses were integrated by Breeze chromatography software
(Version 3.30) (Waters corporation, Milford, MA, USA). Isoﬂavone contents and proﬁles
were determined in all yogurt samples at week one and week six (end of storage at 4 °C).
The eluted isoﬂavones were detected at 262 nm and quantitative data for genistein,
daidzein, genistin and daidzin were obtained from comparison with the known standards.
The stachyose contents of the yogurt samples at ﬁrst week of manufacturing were

evaluated using the HLPC method at ABC Research Corporation (Gainsville, FL, USA).

5.3.5 Statistical Analysis

Statistical analysis was conducted using the statistical function of Microsoft excel
and Sigma plot 9.0 (J andel Scientiﬁc, San Rafael, CA). One-way analysis of variance
using Sigma Stat 3.1 (J andel Scientiﬁc, San Rafael, CA) was conducted, and differences
between the sample means were analyzed by Tukey’s test for multiple comparisons of
means at p < 0.05. Triplicate samples were used for isoﬂavone evaluation while replicate

samples were used for the stachyose evaluation.

5.4 RESULTS AND DISCUSSION

5.4.1 Effects of soy fortiﬁcation on total isoﬂavone content of yogurt

In this study four isoﬂavone isomers evaluated in the yogurt samples were geinstein,
daidzein, genistin and daidzin. The HPLC results for the isomeric isoﬂavones are shown
in Figures 5.1, 5.2 5.3 and 5.4. The respective concentrations of total isoﬂavones in the

yogurt samples at ﬁrst week and sixth week varied over wide ranges depending on the

126

sample. Mean total isoﬂavone concentration varied between 0.00-3 77.15 pg g for 1St
week, and 0-478.66 ug/ g, for 6th week of storage (Table 5.1 and Figures 5.1 and 5.2). The
100% all dairy yogurt (sample 949) did not contain any measurable isoﬂavones (0.00
ug/g) at lSt and 6th week, while 100% all soy yogurt (sample 894) had the highest

concentration at 1St and 6h week (377.15 ug/ g and 478.66 ug/g respectively.

127

Figure 5.1 Representative HPLC chromatogram of isoﬂavones in soy-fortiﬁed yogurts
at 1St week (a, daidzin; b, genistin; c, daidzein; d, genistein). Reverse-phase

 

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128

Figure 5.2 Representative HPLC chromatogram of isoﬂavones in soy-fortiﬁed yogurts
at lSt week(a, daidzin; b, genistin; e, daidzein; d, genistein).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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M15

129

Figure 5.3 Representative HPLC chromatogram of isoﬂavones in soy-fortiﬁed yogurts
at 6th week (a, daidzin; b, genistin; c, daidzein; d, genistein).

 

M); 252 a

 

 

 

 

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(1(1) 2(D 4(1) 6(1) 8(1) 10(1) 1210 14(1) 16(1) 18(1) 21(1) 2(1)
Mm

130

Figure 5.4 Representative HPLC chromatogram of isoﬂavones in soy-fortiﬁed yogurts
at 6th week (a, daidzin; b, genistin; c, daidzein; d, genistein)

 

05) .

 

144 141
99
5"

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131

Table 5.1 Total isoﬂavone concentrations (on dry matter basis) in yogurts fortiﬁed with
germinated or non- germinated soybean powders ( pg/ g) at lst and 6th week of storage
(4°C)

 

 

 

 

 

 

 

 

Yogurt samples 1St Week 6‘” Week Percent increase
I252 131.20 i 13.36CdB 161.30 :l: 8.43“A 18.7
169 149.18 i 18.31°B 230.91 :1: 7.58bA 35.4
344 89.68 i 5.62‘113 128.50 :1: 15.27“A 30.2
159 197.95 :t 26.331’13 259.82 :1‘: 10.26bA 23.8
817 201.951: 24.84b N/A2 N/A
894 377.147 i 6.812113 478.66 :t 27.01aA 21.2
949 0.00 i 0.00“’A 0.00 :1: 0.00‘IA 0.0

 

 

 

 

 

 

a'cMean values in a column with different superscript small letters are Signiﬁcantly

different (p < 0.05); n = 3.

NB Mean values in a row with different superscript capital letters are Signiﬁcantly

different (p < 0.05); n = 3

1252 = 50% germinated Vinton 81 + 50% non-fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk
344 = 50% germinated DF 222 + 50% non-fat dry milk

159 = 50% non-germinated DF 222 + 50% non-fat dry milk
817 = 50% germinated E05276-T + 50% non-fat dry milk
894 = 100% non-germinated Vinton 81 (all soy yogurt- control)
949 = 100% non-fat dry milk (all dairy yogurt- control)
2N/A = Not Available

132

An explanation to the different concentration of isoﬂavones between the samples
could be due to the use of different soybean varieties and differently treated soybean
powders utilized in the yogurt manufacturing. The respective concentration of
isoﬂavones (on dry matter basis) in the yogurts fortiﬁed with non-germinated soy
powders were149.18 i 18.31ug/g (50:50 NGV 81 :NFDM), and 197.95 i 26.33 ug/g
(50:50 NGDF 222:NFDM). On the other hand, the concentration of isoﬁavones in the
yogurt samples fortiﬁed with germinated soy powders at 1 week of manufacturing were
131.2 :t 13.36 ug/g (50:50 GV 81:NFDM), 89.68 i: 5.62 ug/g (50:50 GDF 222:NFDM)
and 201.95 :h 24.84 ug/g (50:50 GETzNFDM). Aside from the control yogurts, the
samples fortiﬁed with germinated E05276-T and non-germinated DF 222 soy powders
had higher (p< 0.05) isoﬂavone contents than the rest. The results obtained in this study
are similar to the data presented by Morandi and others (2005), in their study on the
isoﬂavone content of Italian soy food products, which included soy yogurts.

At the end of 6 weeks of storage, the total isoﬂavone (4 isomers) concentrations in
all the yogurt samples apart ﬁom sample 949 i.e. 100% dairy yogurt, increased
signiﬁcantly (Table 5.2, Figures 5.3 and 5.4). The highest percent increase occurred in
sample 50:50 NGV 81 :NFDM (35.4%), followed by 50:50 GDF 222:NFDM (30.2%),
50:50 NGDF 222:NFDM (23.8%), 100% soy yogurt (21.2%), 50:50 GV 81 :NFDM
(18.7%) and 100% dairy yogurt (0.0%).

Loss of isoﬂavones was determined by Wang and Murphy (1996), during
processing of soybeans into tempeh, tofu, and soy protein isolate. They observed that 61,
44, and 53% of total isoﬂavones were lost during manufacturing of these products

respectively. In our study, we observed a decrease of 26% of total four isoﬂavones during

133

processing of soy powder into yogurt in the 1St week and 6% in 6th week of yogurt

manufacturing using 100% non-germinated Vinton 81 soy powder (Table 5.2).

Table 5.2 Conversion and retention of isoﬂavones (4 isomers) during processing of soy

 

 

 

 

 

 

 

 

powders into yogurts (%)

Yogurt 18‘ Week (%) 1"t Week (%) 6th Week (%) 6‘“ Week (%)
sample Converted Retained Converted Retained
I252 375 62.5 23.2 76.8

169 41.4 58.6 9.3 90.7

344 56.8 43.2 38.0 62.0

159 45.5 54.5 28.4 71.6

817 34.0 66.0 N/A2 N/A

894 26.0 74.0 6.0 94.0

949 00.0 00.0 00.0 00.0

 

 

 

 

 

 

. 1252 = 50% germinated Vinton 81 + 50% non-fat dry milk
169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk
344 = 50% germinated DF 222 + 50% non-fat dry milk

159 = 50% non-germinated DF 222 + 50% non-fat dry milk
817 = 50% germinated E05276-T + 50% non-fat dry milk

894 = 100% non-germinated Vinton 81 (all soy yogurt- control)
949 = 100% non-fat dry milk (all dairy yogurt— control)

2MA = Not Available

134

 

The percentages of isoﬂavones retained in the yogurt samples fortiﬁed with soy
powders were calculated based on the fact that 50% of soy powder was incorporated into
the yogurt base unlike the high percentage retained when 100% soy powder was utilized
for yogurt manufacturing (Table 5.2). This data clearly showed that yogurt
manufacturing process did not adversely affect the isoﬂavone contents of the ﬁnal
product signiﬁcantly. According to Coward and others (1998), certain processing
conditions such as heating including baking and frying did not necessarily alter the total
isoﬂavone contents of soy products but instead changed the proﬁles of individual

isoﬂavones.

5.4.2 Effects of soy fortiﬁcation on Genistein and Genistin content of yogurt
Recent studies have shown that genistein contributed most to the concentration of
aglycones in four soybean extracts fermented with lactic acid bacteria and biﬁdobacteria
(Pyo and others, 2005). The genistein and genistin contents of various soy-fortiﬁed
yogurts at one week and six week of manufacturing are shown in Tables 5 .3 and 5.4.
Previous study showed that 90% of the genistein series was found to be in the conjugated
forms with the B- glucoside (genistin) being higher than the malonyl- or acetylglycoside
forms (Griin and others, 2001). This suggests that only about 10% exists as genistein,
which is similar to our data. Genistein contents of all the yogurt samples were little
affected by storage at 4 0C after 6 weeks because no signiﬁcant increase or decrease was
observed as seen in Table 5.3. The small decreases observed can most likely be attributed

to molecular conversions.

135

Table 5.3 Genistein concentrations in yogurts fortiﬁed with germinated or non-
germinated soybean powders (11 g/ g) at lst and 6th week of storage (4°C)

 

 

 

 

 

 

 

 

Yogurt 1St Week 6til Week % Increase (T)
sample or decreases (l)
1252 11.626 3 2.17dA 9.604 :1: 1.14‘1A 17.4l

169 20.437 :t 0.67"“A 21.336 :1: 0.83bA 5.1 T

344 22.252 a 1.13M 20.432 a 0.80bA 8.2 l

159 17.963 a 1.36‘:A 16.627 2: 0.65"A 7.4 i

817 18.743 :1: 1.37“ N/A2 N/A

894 34 675 i 0.9121A 34.460 :t 0.59“ 0.6 l

949 0.00 :1: 0.00eA 0.00 :1: 0.00“A 0.0

 

 

 

 

 

 

M”Mean values in a column with different superscript small letters are signiﬁcantly
different (p < 0.05); n = 3.
“3 Mean values in a row with different superscript capital letters are signiﬁcantly
different (p < 0.05); n = 3
1252 = 50% germinated Vinton 81 + 50% non-fat dry milk
169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk
344 = 50% germinated DF 222 + 50% non-fat dry milk
159 = 50% non-germinated DF 222 + 50% non-fat dry milk
817 = 50% germinated E05276-T + 50% non-fat dry milk
894 = 100% non-germinated Vinton 81 (all soy yogurt- control)
949 = 100% non-fat dry milk (all dairy yogurt- control)
2N/A = Not Available

136

Table 5.4 Genistin concentrations in yogurts fortiﬁed with germinated or non-
gerrninated soybean powders (ug/ g) at 1st and 6th week of storage (4°C)

 

 

 

 

 

 

 

 

Yogurt lst Week 6tll Week % Increase (T)
sample or decreases (l)
1252 75.552 :1: 7.75bB 95.132 at 398"“ 20.6 T

169 71.662 4: 6.51bB 123.816 a 3.16“ 42-1 T

344 38.895 a 4.56"B 72.440 e 12.47“ 46.3 T

159 79.434 a: 9.841313 120.581 :L- 3.64“ 34.1 T

817 91.527 a 18.68b N/A2 N/A

894 147.971 a 5.798‘13 207.239 :1: 21.85“ 28.6 T

949 0.00 a 0.00“ 0.00 e 0.00“ 0.0

 

 

 

 

 

 

3'6 Mean values in a column with different superscript small letters are Signiﬁcantly

different (p < 0.05); n = 3.

AB Mean values in a row with different superscript capital letters are signiﬁcantly

different (p < 0.05); n = 3

1252 = 50% germinated Vinton 81 + 50% non-fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk
344 = 50% germinated DF 222 + 50% non-fat dry milk

159 = 50% non-germinated DF 222 + 50% non-fat dry milk
817 = 50% germinated E05276-T + 50% non-fat dry milk

894 = 100% non-germinated Vinton 81 (all soy yogurt- control)
949 = 100% non-fat dry milk (all dairy yogurt- control)
2N/A = Not Available

137

Genistin contents were signiﬁcantly affected (p < 0.05) at the end of 6 weeks of
storage. Just like total isoﬂavone contents, the genistin contents at 6 weeks were
signiﬁcantly higher (p < 0.05) in all the yogurt samples that contained soy powders. The
‘ increase in genistin concentration during storage can be attributed to formation of this B-
glucoside by the de—esteriﬁcation of the malonyl- and acetylglycosides (Griin and others,
2001; Coward and others, 1998; Coward and others, 1993). A previous study by Simonne
and others (2000), suggests that the increase could also be due to further hydrolysis of the
malonyl- and acetylglycosides leading to conversion into B-glucoside forms.

As expected, 100% soy yogurt had the highest concentration of genistin. Of the
soy-fortiﬁed yogurts, 50:50 NGDF 222:NFDM and 50:50 NGV 81 :NFDM had the most
genistin contents (Table 5.4). It is worthy to note that these yogurts were made with non-
gerrninated (soaked) soybean powders. Again sample 949 made ﬁom 100%non-fat dry
milk did not contain any genistein or genistin. Some strains of lactic acid bacteria have
been discovered to have B-glucosidase activities whereby they could bioconvert the
glucoside isoﬂavones into their respective aglycones (Chun and others, 2007). In relation
to the metabolism of these isoﬂavones in humans, researches have shown that the
aglycones are more bioactive because they are easily absorbed through the gut wall than
the other isoﬂavone isomers. Thus the need to know the chemical forms in which these
isoﬂavones exist in foods is very important.

5.4.3 Effects of soy fortiﬁcation on Daidzein and Daidzin contents of yogurt

The different concentrations of daidzein and daidzin in soy-fortiﬁed yogurts are

summarized in Tables 5.5 and 5.6. Table 5.5 shows that there was no signiﬁcant

difference between daidzein contents in lst week and 6th week storage at 4 °C.

138

Table 5.5 Daidzein concentrations in yogurts fortiﬁed with germinated or non-
germinated soybean powders ( ug/ g) at 1st and 6th week of storage (4°C)

 

 

 

 

 

 

 

 

Yogurt lSt Week 6til Week % Increase (T)

sample or decreases (l)
252 2.751 a 1.69“ 2.324 a 0.88“ 15.5 i

169 4.039 i 0.3?A 4.766 a 0.15“ 15.3 T

344 4.832 a 0.40“ 4.572 at 0.75“ 5.4 l

159 4.071 a: 0.20“ 4.252 a 0.39“ 4.3 T

817 3.496 d: 0.39b N/A2 N/A

894 12.968 :1: 1.00“ 13.005 a 0.77“ 0.3 T

949 0.00 a 0.00“ 0.00 a 0.00“ 0.0

 

 

 

 

 

 

8'6 Mean values in a column with different superscript small letters are Signiﬁcantly

different (p < 0.05); n = 3.

“3 Mean values in a row with different superscript capital letters are signiﬁcantly

different (p < 0.05); n = 3

1252 = 50% germinated Vinton 81 + 50% non-fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk
344 = 50% germinated DF 222 + 50% non-fat dry milk

159 = 50% non-germinated DF 222 + 50% non-fat dry milk
817 = 50% germinated E05276-T + 50% non-fat dry milk
894 = 100% non-germinated Vinton 81 (all soy yogurt- control)
949 = 100% non-fat dry milk (all dairy yogurt- control)
2N/A = Not Available

139

The observation on daidzein contents made in the above table is similar to that
made in Table 5.3, that showed no signiﬁcant difference in genistein contents of freshly
manufactured soy-fortiﬁed yogurts and six week old soy-fortiﬁed yogurts. The highest
daidzein content was found in 100% soy yogurt (sample 894). There were no statistically
signiﬁcant differences between all the soy-fortiﬁed yogurts despite the differences in
soybean variety and treatment. The overall low amounts of daidzein across the samples
when compared to genistein, are consistent with the ﬁndings of other workers on the
daidzein and genistein contents in soy foods (Preinerstorfer and Sontag, 2004; Morandi
and others, 2005). Daidzein is a very unstable isoﬂavone, thus its low concentrations in
soy foods could be attributed to this instability as a result of bioconversion to other forms
of isoﬂavones (Coward and others, 1993; Coward and others, 1998; Mahungu and others,
1999; Griin and others 2001).

As a result of the health beneﬁts attached to the consumption of isoﬂavones, the
chemical forms in which they appear in foods is considered important since it can
dramatically inﬂuence the biological activity, the bioavailability, and therefore the
physiological effects of these dietary constituents. After ingestion, soybean B-glucosides
are hydrolyzed by intestinal glucosidases, which release the aglycones, daidzein,
genistein and glycitein (Chun and others, 2007). These could be absorbed directly or
further metabolized to many speciﬁc metabolites, including equol and p-ethylphenol
(3,4). Genistin and daidzin contents again were more abundant than genistein and
daidzein contents (Figure 5.4). Ingestion of a diet rich in isoﬂavone-aglycones may be

more effective in increasing health beneﬁts associated with soybean consumption.

140

Table 5.6 Daidzin concentrations in yogurts fortiﬁed with germinated or non-

germinated soybean powders (ug/ g) at 1st and 6th week of storage (4°C)

 

 

 

 

 

 

 

 

 

 

 

 

Yogurt sample lSt Week 6‘“ Week % Increase (T)
or decreases
(l)

1252 41.267 :1: 500°“ 54.237 :1: 4.07“ 23.9 T

169 53.037 3: 11.79"B 80.991 a 5.80“ 34.5 T

344 23.700 t 8.09dB 31.059 at 5.27“ 23.7 T

159 96.484 4 25.73“ 118.355 a 11.25“ 18.5 T

817 87.830 at 19.6?Sc N/A2 N/A

894 181.527 a 7.31“ 232.817 3: 17.12“ 22.0 T

949 0.00 :1: 0.00“ 0.00 :1: 0.00“ 0.0

 

8'6 Mean values in a column with different superscript small letters are signiﬁcantly

different (p < 0.05); n = 3

M3 Mean values in a row with different superscript capital letters are signiﬁcantly
different (p < 0.05); n = 3
1252 = 50% germinated Vinton 81 + 50% non-fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk

344 = 50% germinated DF 222 + 50% non-fat dry milk
159 = 50% non-germinated DF 222 + 50% non-fat dry milk
817 = 50% germinated E05276-T + 50% non-fat dry milk

894 = 100% non-germinated Vinton 81 (all soy yogurt- control)

949 = 100% non-fat dry milk (all dairy yogurt- control)
2N/A = Not Available

141

 

Figure 5.5 Isoﬂavone concentrations in 1 week (A) and 6 week (B) old yogurt samples

 

 

 

 

 

 

 

 

 

 

 

A
300 -
250 i - #252
15:3 #169
a, 200 - _ #344
‘ I:
g #159
6 150 ~
c
.c
'i 100 -
50 ..
o _
Genistein Daidzein Genistin Daidzin
B
300
250 .
- #252
U) 200 ~ E22} #169
~ — #344
3 1:1 #159
5 150 7 — #894
r:
o - #949 _ '1'
o 100
50
o .. .. L.

 

 

 

 

 

Genistein Daidzein Genistin Daidzin

252 = 50% germinated Vinton 81 + 50% non-fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk
344 = 50% germinated DF 222 + 50% non-fat dry milk

159 = 50% non-germinated DF 222 + 50% non-fat dry milk
817 = 50% germinated E05276-T + 50% non-fat dry milk

894 = 100% non-germinated Vinton 81 (all soy yogurt- control)
949 = 100% non-fat dry milk (all dairy yogurt- control)

142

5.4.4 Effects of soy fortiﬁcation on Stachyose content of yogurt

The stachyose contents of all the soy samples after one week of storage are

summarized in Table 5.7.

Table 5.7 Stachyose contents of yogurts fortiﬁed with germinated or non-germinated

whole soy powders after l-week storage at 4 °C.

 

 

 

 

 

 

 

 

Treatment (Yogurt samples Stachyose (mg/g)
1252 4.41 a 0.19d
169 10.04 a 0.23b
344 3.40 1 0.11e
159 8.45 :l: 0.07“
817 2.82 a 0.13f
894 17.25 a 0.073
949 1.33 a 0.01g

 

 

 

 

a'g Mean values in a column with different superscript small letters are signiﬁcantly
different (p < 0.05); n = 2

1252 = 50% germinated Vinton 81 + 50% non-fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk

344 = 50% germinated DF 222 + 50% non-fat dry milk

159 = 50% non-germinated DF 222 + 50% non-fat dry milk

817 = 50% germinated E05276-T + 50% non-fat dry milk

894 = 100% non-germinated Vinton 81 (all soy yogurt- control)

949 = 100% non-fat dry milk (all dairy yogurt- control)

143

Table 5.7 shows that stachyose content in each yogurt was signiﬁcantly different
(p < 0.05) from each other. Overall yogurts made with either 50% or 100% non-
germinated soy powders had higher stachyose contents irrespective of the soybean
variety. Sample 894 i.e. all soy yogurt made with non-germinated Vinton 81 contained
the highest amount of stachyose ( l 7 .25 mg/g). This was followed by sample 169 (50%
non-germinated Vinton 81) with 10.04 mg/ g of stachyose and sample 159 (50% non-
germinated DF 222) with 8.45 mg/ g of stachyose. Sample 949 (all dairy yogurt) had
negligible amount of the compound, which was attributed to interference from sucrose
since non-fat dry milk does not contain stachyose. Overall all the yogurt samples
prepared with germinated soy powders had lower quantities of stachyose. This
observation conﬁrms earlier ﬁndings that oligosaccharide contents are reduced during
germination due to increase in a-galactosidase activity (V iana and others, 2005) and
ﬁrrther reduced during fermentation by lactic acid bacteria.

Table 5.8 shows the percent reduction in stachyose in yogurt samples ﬁom the base
soy powders. The stachyose content of all the soy-fortiﬁed yogurts made with 50% of soy
powder were highly reduced and ranged ﬁom 51.5 to 63.8% reduction in the yogurts.
Also yogurt made with 100% non-germinated soy powder (i.e. sample 894) had 58.3%
reduction in stachyose content. Chen and others (2004) reported that amino acids and
organic acids concentrations could be increased by the increase in the activity and
viability of probiotics due to the presence of prebiotics in milk to be used for yogurt
manufacturing. A diet high in non-digestible carbohydrate causes increased intestinal
fermentation and could result in more extensive biotransformation of phytoestrogens,

leading to increased formation of equol, a mammalian isoﬂavone metabolite. Equol is

144

said to have estrogenic potency of magnitude higher than that of its plant precursor,

daidzein (Joannou and others, 1995).

Table 5.8 Percent reduction of stachyose in yogurts manufactured with germinated and

non-germinated soy powders

 

 

 

 

 

 

 

 

Soy powder Yogurt sample % Stachyose reduced
1CV 81 (9.58 mg/g) 2252 (4.41 mg/g) 53.9
NGV 81 (20.68 mg/g) 169 (10.04 mg/g) 51.5
NGV 81 (41.35 mg/g) 894 (17.25 nag/g) 58.3
GDF 222 (7.73 mg/g) 344 (3.40 mg/g) 56.0
NGDF 222 (23.33 mg/g) 159 (8.45 mg/g) 63.8
GET (6.95 mg/g) 817 (2.82 mg/g) 59.4

 

 

 

 

lGV 81 = Germinated Vinton 81

NGV 81 = Non-germinated Vinton 81

GDF 222 = Germinated DF 222

NGDF 222 = Non-germinated DF 222

GET = Germinated E05276-T

2252 = 50% germinated Vinton 81 + 50% non-fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk
344 = 50% germinated DF 222 + 50% non-fat dry rrrilk

159 = 50% non-germinated DF 222 + 50% non-fat dry milk
817 = 50% germinated E05276-T + 50% non-fat dry milk

894 = 100% non-germinated Vinton 81 (all soy yogurt- control)

145

As reported by several workers, consumption of oligosaccharides could lead to
improved immune and nutritional status as well as reduced risk of Crohn’s disease, colon
cancer, irritable bowel syndrome etc. (Gibson and Angus, 1996; Sako and others, 1999).
In summary, the data obtained in this objective indicate that each of these soy-fortiﬁed
cow milk based yogurts contain the following amount of isoﬂavone (4 isomers) per
serving (about 200 ml). On a dry matter basis, sample 252 (germinated Vinton 81 +
NFDM) had 26.2 mg/serving; 169 (non-germinated Vinton 81 + NFDM) had 29.8
mg/serving; 344 (germinated DF 222 + NFDM) had 17.9 mg/serving; 159 non-
gerrrrinated DF 222 + NFDM) had 39.6 mg/serving; 817 (gerrrrinated E05276-T +
NFDM) has 40.4 mg/serving; 894 (non-germinated Vinton 81 only) had 75 mg/serving.
Suggested amount of total isoﬂavones (12 isomers) per serving is 40-50 mg. Also
suggested amount of dietary ﬁber per serving is between 3-5 grams per serving. Our
study showed that the stachyose content in the yogurt samples varied ﬁom 0.6 g/ serving

for germinated E05276-T + NFDM to 3.45 g/serving for 100% soy yogurt.

146

CHAPTER 6

SHELF LIFE STUDIES AND VIABILITY OF WHOLE SOY-FORTIFIED

YOGURTS STORED AT 4 0C

6.1 ABSTRACT

Fermented dairy foods produced with probiotic bacteria have attracted a lot of
research interest because of their potential health beneﬁts. The growth and viability of
lactic acid bacteria and probiotic used in making whole soy-fortiﬁed yogurts were
monitored at 7-day interval during 6 weeks of storage at refrigeration temperature (4 °C).
MRS, MRS-sorbitol and M17 agar were utilized to enumerate Lactobacillus delbreuckii
subsp. bulgaricus, Lactobacillus acidophilus NCFM and Streptococcus thermophilus
respectively. The pH of each yogurt was also measured on a weekly basis during storage.
The growth and viability of all the cultures were above 10 7 CFU/ g of yogurt. The lowest
concentrations of all the cultures were obtained in 100% whole soy yogurt. Alternatively,
the highest cell concentrations occurred in whole soy—fortiﬁed yogurts especially in the
sample made with germinated DF 222 soy powder.

Overall the pH values of each yogurt sample remained constant up to the 5th week
of cold storage. The pH values of the sample with 100% non-germinated soy powder
were signiﬁcantly higher than the rest of the samples, ranging from 4.67 for 1St week to
4.82 for 6th week. In general, there was no statistically signiﬁcant difference in pH
between germinated DF 222 soy-fortiﬁed yogurt and 100% dairy yogurt sample during

storage. There was a signiﬁcant difference in pH values between lit and 2nd week of

147

storage and between 31rd and 4th weeks of storage among the samples. The pH values of
whole soy-fortiﬁed including the control yogurts were between 4.5 and 4.8 after 6 weeks

of storage at 4 °C.

6.2 INTRODUCTION

There has been a world increase in the consumption of fermented dairy product
containing probiotics. Probiotics are deﬁned as “live microorganisms that when
administered in adequate amounts confer a health beneﬁt on the host (FAO, 2002). These
probiotics were initially isolated from human sources and their health beneﬁts have been
a focus of intense research internationally (O’Sullivan and others 1992; Ostlie and others
2003). Several research studies have shown the health beneﬁts of some the well-
characterized lactic acid bacteria (Salminen and others, 1996; Saarela and others 2000). It
has been suggested that in order for a microorganism to exert positive health beneﬁts, the
microbial concentration must be at least 106 CF U/ g in the product throughout the shelf
life period of the product (Vinderola and others 2000, 2002; Fanworth, 2005).

Traditionally, yogurt is manufactured by the addition of two starter cultures,
Lactobacillus delbruekii subsp. bulgaricus and Streptococcus thermophilus to milk
(Tammirne and Marshall 1997). Subsequent fermentation of milk as a result of the starter
culture metabolism leads to the taste and bioactive metabolites, which contribute to the
health promoting properties of yogurt (Rachid and others 2002; Donkor and others 2005).
Dairy yogurt is widely consumed in both the developed and developing countries, but
recently there is a demand to alternative to cow’s milk due to problems with allergenicity,

desire for vegetarian alternatives, insufﬁcient production of dairy milk, and new products.

148

Hence a keen interest in soymilk-based yogurts has developed although mostly probiotic
cow’s milk based yogurts are currently being marketed (Lee and others 1990; Nsofor and
others 1992,1996).

Probiotics have been implicated in the treatment of different types of diseases such
as diarrhea (Sarker and others, 2005; Szymansky and others, 2006), Crohn’s disease
(Bousvaros and others, 2005), and urogenital infections (Reid and others, 2003). Yogurt
is considered a good vehicle for introducing probiotics to consumers. It is now popular to
use probiotics such as Lactobacillus sp and Biﬁdobacterium as adjunct starter cultures
(Dave and Shah, 1998; Gardiner and others, 2002; Shah 2004). Several studies have
shown that after probiotic ingestion, biological barriers such as stomach acid and bile
prevent the survival of these probiotics in the intestinal tract (Lankaputhra and Shah,
1995) therefore their ability to confer health beneﬁts is impeded. Also milk has been
shown to be a poor medium for the growth and survival of probiotics because milk does
not contain enough amino acids and lower molecular weight peptides to sustain such
growth (Shah, 2006).

In order to maintain the presence of these probiotics in large numbers in the gut,
many workers have supplemented the milk base with various compounds such as soy
protein isolate (Pham and Shah, 2008), soy protein concentrate (Drake and others, 2000;
Drake and Gerard, 2003), ﬁbers (F emandez-Garcia and others, 1998; Aryana and others,
2007). Prebiotics are non-digestible carbohydrates that are not easily hydrolyzed or
digested in the upper part of the gastrointestinal tract but can be metabolized by some
probiotics. lrrulin and oligosaccharides are known to enhance the activities and growth of

probiotics such as Bifidobacterium spp (Shin and others 2000) and Lactobacillus

149

acidophilus (Aryana and others, 2007). In 2004, Chen and others discovered that the
concentrations of organic acids and arrrino acids can be increased in yogurts containing
added prebiotics that could in turn increase the activities of the probiotics. Research
evidence shows that incorporation of prebiotics to yogurt containg Lactobacillus
acidophilus would probably lead to a healthier yogurt. The objective of this study was to
evaluate the shelf life of yogurts fortiﬁed with germinated and non-germinated whole soy
powders from various soybean varieties and fermented with traditional yogurt cultures
and a probiotic culture, Lactobacillus acidophilus (La NCFM). The whole soy powders
utilized contain oligosaccharides e.g. stachyose that could serve as a prebiotic, thereby

eliminating supplementation of the yogurt base with extraneous ﬁbers.

6.3 MATERIALS AND METHODS

The viability of Lactobacillus delbruekii subsp. bulgaricus, Streptococcus
salivarius subsp. thermophilus and Lactobacillus acidophilus (NCFM) used in making
whole soy-fortiﬁed yogurts were monitored at 7-day interval during 42 days of storage.
The yogurt samples were stored in the refrigerator (4 °C) throughout the study and these
samples belonged to the same batch of yogurts utilized for the sensory evaluations and

chemical analysis.

6.3.1 Media Preparation
Diluents of peptone (0.1%) and water were prepared by dissolving one gram of
bacto-peptone medium (Difco Laboratories, Detroit, MI) in one liter of distilled water

and sterilized by autoclaving at 121 °C for 15 minutes. In order to enumerate S. salivarius

150

subsp. thermophilus, M17 agar was prepared. M17 agar was prepared according to
manufacturer’s instructions by adding 37.25 g of M17 broth powder (Difco Laboratories,
Detroit, MI), 1.5% bacto agar (Difco Laboratories, Detroit, MI) to 1 L of distilled water
and also sterilized at 121 °C for 15 min., before, added 50 ml 10% sterile lactose solution,
pouring into sterile agar plates. De Man, Rogosa, Sharpe (MRS) agar was used to
enumerate Lactobacillus delbruekii subsp. bulgaricus. This agar was prepared by adding
70 g of MRS agar (Difco Laboratories, Detroit, M1) to 1 L of distilled water, sterilized at
121 °C for 15 min., before pouring into agar plates. Modiﬁed MRS agar was utilized to
enumerate Lactobacillus acidophilus NCFM. This agar was prepared by adding 10%
ﬁlter sterile sorbitol to sterilized dextrose free MRS agar (1% ﬁnal sorbitol concentration

in medium) before pouring on agar plate (Dave and Shah, 1996).

6.3.2 Enumeration of Lactic Acid Bacteria

The following yogurt samples were utilized for shelf life studies; 50% non-
i germinated DF 222 + 50% non-fat dry milk, 50% germinated DF 222 + 50% non-fat dry
milk, 50% non-germinated + 50% non-fat dry milk, 50% germinated Vinton 81 + 50%
non-fat dry milk, 100% non-germinated Vinton 81- all soy control and 100% non-fat dry
milk- all dairy control. Starting at 0 week, one gram of sample was taken from each
yogurt and diluted with 99 ml of sterile 0.1% (w/v) peptone and suitable serial dilutions
were plated on each of the selective medium prepared (M17, MRS, MRS-sorbitol) in
triplicates. Serially diluted tubes of 10‘5 were utilized for plating on agar plates.

The diluted yogurt samples plated on M17 agar for the enumeration of S.

salivarius subsp. thermophilus were incubated aerobically at 37 °C for 24h. Also samples

151

plated on MRS and MRS-sorbitol agars were incubated anaerobically using Gas Packs
(BBL Microbiology Systems, Cockeysville, MD) at 37 °C for 24h. Plates containing 25-
250 colonies were enumerated and recorded as colony—forming units per gram (CF U/ g) of
culture. The colonies were counted using 920A colony counter (American Bantex Corp.,
Burlingarne, CA). The pH of each yogurt sample was also measured in replicate at 7-day

interval for 42 days (shelf life period).

6.3.3 Statistical analysis

Bacterial enumeration was done in triplicate while pH monitoring was carried out
using replicate samples. Fixed effects for the viability and pH of the cultures included
two factors i.e. yogurt sample and storage time (weeks). Two-way analysis of variance
was done to evaluate these effects and their interactions. Sigma Stat 3.1 and Sigma plots
9.0 (Jandel Scientiﬁc, San Rafael, CA) were used for this analysis and Tukey’s test was
used for comparisons of means. Comparisons were considered signiﬁcantly different if p

< 0.05.

6.4 RESULTS AND DISCUSSION
6.4.1 Viability of microorganisms in yogurts during cold storage

The effects of whole soy powder supplementation for milk-based yogurt on the
viability of lactic cultures throughout 42 days of refrigerated storage were studied. An
important parameter in evaluating viable organisms during shelf studies is the ability to

differentiate each bacteria used.

152

6. 4. 1. I Viability of Lactobacillus delbreuckii subsp.bulgaricus during cold storage
Table 6.1 shows the analysis of variance (ANOVA) for the independent variables
i.e. yogurt samples containing Lactobacillus delbreuckii subsp.bulgaricus and weeks (0,

1, 2, 3, 4, 5, and 6) and their interactions on MRS agar.

Table 6.1 Analysis of variance for the effects of yogurt varieties and storage time
(weeks) on the viability of Lactobacillus delbreuckii subsp.bulgaricus (CFU/ g)

 

 

 

 

 

 

 

Main DF ss Ms F P
Effects

Yogurt 5 2.291 x1016 4.582 x1015 28.158 <0.001
samples

Weeks 5 9.948 x10” 1.990 x10” 12.228 <0.001
Yogurt x 25 1.14 x 1016 4.569 x10” 2.808 <0.001
Weeks

Residual 72 1.172 x10"5 1.627 x10”

Total 107 5.599 x10l6 5.233 x10”

 

 

 

 

 

 

 

The above table suggests that the difference in the mean values among the
different varieties of yogurt samples was higher and more signiﬁcant with an F-value of
28.158 (p < 0.001), than the effects of differences in weeks (F-value =12.228, p < 0.001).
The two-way analysis of variance also suggests that the effect of different varieties of
yogurt samples depends on storage time, hence there was a statistically signiﬁcant
interaction between yogurt samples and weeks (p < 0.001). Based on this observation,
one-way analysis of variance was then used to compare the means (CF U/ g) of yogurt
samples per week (Figure 6.1). On the lSt week of storage, there was no signiﬁcant

difference between 100% dairy yogurt and all the 50% soy-fortiﬁed yogurts. All these

153

samples were signiﬁcantly different from 100% soy yogurt, which had the lowest count
of L. delbrueckii subsp.bulgaricus.

The viable cell counts were between 2.3 x 107 CFU/g and 5.9 x 107 CFU/g
initially. L. delbrueckii subsp.bulgaricus attained or maintained viable cell numbers after
6 weeks of cold storage (2.1 x 107 CFU/g to 9.1 x 107 CFU/g). All the yogurt samples
consistently maintained high cell counts up until week 5. By week 6 few of the cell
counts decreased but the cell numbers were still high enough for the recommended
106 CFU/ g or greater for health claims (Ouwehand and Salminerr, 1998). Growth of L.
delbrueckii subsp.bulgaricus was slower in 100% soy yogurt than in 100% dairy yogurt
but later increased by the 2nd to San weeks of storage. The improved growth during this
period could be as result of pH drop of the soy yogurt during cold storage, which favors
the growth of lactobacilli generally (Farnworth and others, 2007). Overall, the soy
powder fortiﬁed samples maintained viability better than the control yogurt samples and
germinated DF 222 fortiﬁed yogurt consistently maintained the highest CFU value
throughout storage.

Differences in the viability of L. delbrueckii subsp.bulgaricus within each sample
during 6 weeks of cold storage are shown in Table 6.2. The data revealed that there was
no signiﬁcant difference in viable cell counts during storage time in yogurt samples
prepared with 50% non-germinated DF 222 and 100% non-fat dry milk. The remaining
yogurt samples showed slight signiﬁcant differences in CFU per week (increase or
decrease) although the cell numbers still remained high. The 100% soy yogurt (control
sample) contained the least number of viable cells especially in the Iii and 6th weeks of

storage (2.3 x 107 and 2.1 x 107 CFU/ g respectively).

154

Figure 6.1 Viability counts of Lactobacillus delbreuckii subsp.bulgaricus (CFU/ g) ﬁom
germinated and non-germinated whole soy-fortiﬁed yogurt samples during 6 week

storage.

1 .4e+8 -

 

1.28+8 -' + C

1 .Oe+8 -

 

 

 

8.0e+7 d

CF U/g

6.0e+7 -

 

 

 

0.0 l 1 l l I 1 l
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

Storage at 4 °C

A (252) = 50% germinated Vinton 81 + 50% non-fat dry milk

B (l 69)= 50% non-germinated Vinton 81 + 50% non-fat dry milk
C (344) = 50% germinated DF 222 + 50% non-fat dry milk

D (159) = 50% non-germinated DF 222 + 50% non-fat dry milk

E (894) = 100% non-germinated Vinton 81 (all soy yogurt- control)
F (949) = 100% non-fat dry milk (all dairy yogurt- control)

155

Table 6.2 Viability of Lactobacillus delbreuckii subsp.bulgaricus (CF U/ g) during six

weeks of storage at 4 °C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Weeks *252 169 344 159 894 949

of

storage

1 5.57x107b 4.47x107b 5.88x107c 5.47x107a 2.27x107bc 5.01x107a
2 6.31x107b 7.49x107“ 7.90x107b 4.61 x107a 4.45x107“ 6.69 x10721
3 6.73x107“ 6.80x107ab 1.02x10“b 5.63 x107a 4.60x107a 6.37 x10721
4 8.27x107“ 7.77x107a 1.17x1088 8.26x107a 4.17x107“ 6.43 x107111
5 9.50x107a 8.00x107a 9.90x107“ 7.00x107a 4.60 x10721 6.60 x107a
6 6.47x107b 9.07x107a 8.37x107bc 6.57x107" 2.13 km“ 5.87x1'07“

 

a'c Mean values in a column with different superscript small letters are signiﬁcantly
different (p < 0.05); n = 3.
*252 = 50% germinated Vinton 81 + 50% non-fat dry rrrilk
169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk
344 = 50% germinated DF 222 + 50% non-fat dry milk

159 = 50% non-germinated DF 222 + 50% non-fat dry milk

894 = 100% non-germinated Vinton 81 (all soy yogurt— control)

949 = 100% non-fat dry milk (all dairy yogurt- control)

156

 

6. 4. 1.2 Viability of Streptococcus thermophilus during cold storage

Streptococcus thermophilus cultures were enumerated on M17 agar and incubated
aerobically at 37 0C for 24-48h. The differences in the mean values among the treatment
groups i.e. yogurt samples and weeks of storage were greater than would be expected by
chance; there is a statistically signiﬁcant difference (p = 0.002). All the soy-fortiﬁed
yogurt samples contained the highest number of S. thermophilus and were statistically
signiﬁcant from the two control samples i.e. all dairy yogurt and all soy yogurt in the ﬁrst
week. This could be attributed to the synergistic effect produced by the two yogurt bases
i.e. milk powder and soy powder. During increased storage to six weeks, the cultures
increased both in grth and viability in all the yogurts but consistently the two control
samples remained the lowest. Overall, the sample containing 100% soy powder, had the
lowest cell counts throughout the shelf life study ranging from 2.98 x 107 in the 1St week,
then increased to 4.83 x 107 in the 3rd week and decreased to 3.00 x 107 in the last week in
the refrigerator.

In general, S. thermophilus showed little or no signiﬁcant change (p < 0.05)
during storage in cell counts from week 1 to week 6 in all the samples ( Figure 6.2 and
Table 6.3). In most cases, the cell concentrations of the yogurt cultures in the soy-
fortiﬁed yogurts were signiﬁcantly (p < 0.05) higher compared with that assessed in the
control dairy and soy yogurts. These results agreed with similar results obtained by

Donkor and others (2005).

157

Figure 6.2 Viability counts of Streptococcus thermophlilus (CF U/g) from germinated
and non-germinated Whole soy-fortiﬁed yogurt samples during 6 week storage

 

 

 

 

1.2e+8 -
+ A
—o— B
1.0e+8 - + C ‘
+ D u
+ E . ’\
8.0e+7 4 ‘D— F v \ /
' / . «? --

6.0e+7 .

CF Ulg
t"
g
1'

 

 

4.0e+7 -
I
I
2.0e+7 4
0.0 ﬁ ﬁ l T I I

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

Storage at 4 °C

A (252) = 50% germinated Vinton 81 + 50% non-fat dry milk

B (169) = 50% non-germinated Vinton 81 + 50% non-fat dry rrrilk
C (344) = 50% germinated DF 222 + 50% non-fat dry milk

D (159) = 50% non-germinated DF 222 + 50% non-fat dry rrrilk

E (894) = 100% non-germinated Vinton 81 (all soy yogurt- control)
F (949) = 100% non-fat dry milk (all dairy yogurt- control)

158

Table 6.3 Viability of Streptococcus thermophilus (CFU/ g) during six weeks of storage

 

 

 

 

 

 

 

at4°C

Weeks #252 169 344 159 894 949

of

storage

1 6.97x107“ 6.71x107“ 6.80x107c 6.03 x107a 2.98x107a 2.39x107b
2 5.49x107b 6.82x107ab 6.87x107b" 6.04x107a 3.69x107a 7.35::107a
3 8.63x107“ 5.60x107b 1.03x108a 8.50x107a 4.83 x107a 6.83 x1073
4 8.83x107a 9.27x107a 9.93x107“ 6.53x107a 3.37x107a 6.33 x10721
5 7.63x107ab 6.97x107“ 8.80x107bc 6.53 x10721 3.83 x107a 6.07x1078
6 6.80x107“ 8.20x107“ 1.00x108 8.10x107a 3.00x107a 7.07 x107a

 

 

 

 

 

 

 

 

a-c

different (p < 0.05); n = 3.
*252 = 50% germinated Vinton 81 + 50% non—fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk
344 = 50% germinated DF 222 + 50% non-fat dry milk
159 = 50% non-germinated DF 222 + 50% non-fat dry milk

894 = 100% non-germinated Vinton 81 (all soy yogurt- control)

949 = 100% non-fat dry milk (all dairy yogurt- control)

159

Mean values in a column with different superscript small letters are signiﬁcantly

 

6.4.1.3 Viability of Lactobacillus acidophilus during cold storage

The probiotic strain Lactobacillus acidophilus (La NCFM) grew well in soy-
fortiﬁed yogurts reaching the health claim levels above 106 CF U/g. Figure 6.3 and Table
6.4 show the viable microbial number (CFU/ g) during cold storage at 4 °C. As indicated,
100% soy yogurt had the lowest cell count although the counts increased steadily from
1.97 x 107 to 4.17 x 107 CFU/g during 6 weeks of storage. This observation is similar to
the previous results above obtained with Streptococcus thermophilus and Lactobacillus
delbreuckii subsp.bulgaricus cultures in the same yogurt sample (made with 100% soy).
This then conﬁrms the ﬁndings in the culture activity studies Where the least activities
and growth were obtained with 12% reconstituted non-germinated soy powder. Similarly,
growth and viable counts of L. acidophilus (La NCFM) in 100% dairy yogurt also were
slightly low ranging from 3.72 x 107 to 5.07 x 107 CFU/g in 6 weeks during storage.

The yogurt fortiﬁed with DF 222 germinated soy powder had the highest growth
and viability throughout the 6 weeks of shelf life studies, followed by the other soy-
fortiﬁed yogurts (Figure 6.3). This data indicate that there were more available nutrients
in the yogurt samples to sustain the increased growth and viability of the probiotic despite
the presence of the other cultures. Previous studies have indicated poor growth and
viability of probiotic cultures when grown together with traditional lactic acid bacteria
used for fermented foods (Vinderola and others, 2002; Sodini and others, 2002). Our data
suggest that all the bacteria utilized in the yogurt making were able to survive in the

presence of one another.

160

Figure 6.3 Growth and viability counts of Lactobacillus acidophilus NCFM (CFU/g)
ﬁ'om germinated and non-germinated whole soy-fortiﬁed yogurt samples during 6-week

 

 

 

 

 

 

storage
1.2e+8 -
+ A
-O— B
1.0e+8 - + C
—A— D ._
E [‘0
AZ. A
a /0
a 6.0e+7 - [ii/j .
I
4.0e+7 - :/
2.0e+7 -
0.0 T l l I I f
Week1 Week2 Week3 Week4 Week5 Week6
Storage at 4 °C

A (252) = 50% germinated Vinton 81 + 50% non-fat dry milk

B (169) = 50% non-germinated Vinton 81 + 50% non-fat dry milk
C (344) = 50% germinated DF 222 + 50% non-fat dry milk

D (159) = 50% non-germinated DF 222 + 50% non-fat dry milk

E (894) = 100% non-germinated Vinton 81 (all soy yogurt- control)
F (949) = 100% non-fat dry milk (all dairy yogurt- control

161

Table 6.4 shows the difference in growth and viability of La NCFM within each yogurt
sample stored in the reﬁ'igerator for 6 weeks. The CFU/ g of all-dairy yogurt sample (F)
was statistically the same throughout the shelf life period of 6 weeks, while the CFU/ g of
soy-fortiﬁed yogurts generally increased aﬁer 1 week of storage. The probiotic culture
grew well in the soy-fortiﬁed yogurts during storage. There was a signiﬁcant increase (p
< 0.05) in cell counts between the 1St week and 6tltl week of storage in yogurts containing

soy powders (Table 6.4) irrespective of soybean treatment or soybean variety.

162

Table 6.4 Viability of Lactobacillus acidophilus NCFM (CFU/ g) during six weeks of

 

 

 

 

 

 

 

storage at 4 °C

Weeks *252 169 344 159 894 949
of

storage

1 4.69x107b 3.48on7b 4.71 x107“ 4.21 x107“ 1.97x107c 3.72x10781
2 5.39x107ab 6.31x1o7a 5.79x1o7bc 5.6Ox107bc 2.92x107bc 5.01x1073
3 6.87x107ab 6.73x107a 6.97x107b 7.07x107‘“b 2.40x107b° 5.23;:107a
4 7.73x107ab 7.06x107a 8.50x107ab 8.03 x1078 3.60x107abc 4.73x107a
5 8.80x107a 7.63x107a 9.47x107a 7.83x1075" 4.872007“ 5.80xlo7a
6 7.43x107ﬁ’ 8.43 x167a 9.43x107a 7.00on7 4.17x107ab 5.07x107le

 

 

 

 

 

 

 

 

3": Mean values in a column with different superscript small letters are signiﬁcantly
different (p < 0.05); n = 3.
*252 = 50% germinated Vinton 81 + 50% non-fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk

344 = 50% germinated DF 222 + 50% non-fat dry milk

159 = 50% non-germinated DF 222 + 50% non-fat dry milk
894 = 100% non-germinated Vinton 81 (all soy yogurt- control)
949 = 100% non-fat dry milk (all dairy yogurt- control)

163

 

6.4.2 pH changes of yogurt samples during cold storage

The pH changes of all the yogurt samples appeared to be dependent on the type of
yogurt and storage period (Table 6.5) .The F-value of 772.436 at p < 0.001 indicate that
the difference in the mean values among the different levels of yogurt samples is

statistically signiﬁcant.

Table 6.5 Analysis of variance for the effects of yogurt varieties and storage time

 

 

 

 

 

 

(weeks) on pH
Main DF SS MS F P
Effects
Yogurt 5 0.826 0.165 772.432 <0.001
samples
Weeks 5 0.205 0.0411 192.135 <0.001 -
Yogurt x 25 0.175 0.00700 32.741 <0.001
Weeks
Residual 36 0.00770 0.000214
Total 71 1.214 0.0171

 

 

 

 

 

 

 

 

The above table also indicates that the difference in the mean values among the different
levels of weeks is statistically signiﬁcant with an F -value of 192.135 (p < 0.001). There is
statistically signiﬁcant interaction between the different types of yogurt and storage time
(F-value = 32.741; p < 0.001). The pH values of the sample with 100% non-germinated
soy powder were signiﬁcantly higher than the rest of the samples, ranging from 4.67 for
1St week to 4.82 for 6th week (Table 6.6). This observation is consistent with our previous
study on the activity of lactic acid bacteria. Overall, there was no statistically signiﬁcant

difference in pH between germinated DF 222 soy-fortiﬁed yogurt and 100% dairy yogurt

164

during storage. Also there was signiﬁcant difference in pH values between 1st and 2nd
week of storage and between 3rd and 4th weeks of storage (Table 6.6).

By the end of storage period, the pH of all products was higher than that recorded
at the termination of yogurt incubation. These increases in pH could be as a result of
proteolytic activities of the cultures including the probiotic during prolonged cold
storage, which resulted to higher levels of liberated amino groups (Nielsen and others,
2001; Donkor and others, 2005). These data were in contrast to the data obtained by
Lamoureux and others (2002) and Popa (2005), whose data showed that there were slight
decrease in yogurt pH during the 42 days of storage at 4 °C, which probably led to lower
cell counts (CFU/ g). The pH recommended for the survival of some probiotics e. g.
biﬁdobacteria in yogurts is 4.6 (Shah, 1996). The pH values of our whole soy-fortiﬁed

including the control yogurts were between 4.5 and 4.8 after 42 days of storage at 4 °C.

165

Table 6.6 pH of soy—fortiﬁed yogurt samples during prolonged cold storage at 4 °C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Weeks of *252 169 344 159 894 949

storage
1 4.56bC 4.59"“C 4.39“ 4.492113 4.67aﬂD 4.41“A
2 4.56ac 460"“: 4.34aA 4.4933 4.69“” 4.372116‘
3 4.562113 4.62ac 4.46CA 4.64bC 4.75”) 4.47"A
4 4.645113 4.6733“ 4.437”A 4.62bC 484°" 4.46bA
5 4.56ac 4.6381) 4.375le 4.60bCD 4.76bE 4.44abB
6 4.67bB 4.79bC 4.68dB 4.49aA 482°C 455°A

a-c; A-E

Comparisons are made within the same column

(H)

252 = 50% germinated Vinton 81 + 50% non-fat dry milk

169 = 50% non-germinated Vinton 81 + 50% non-fat dry milk

344 = 50% germinated DF 222 + 50% non-fat dry milk

159 = 50% non-germinated DF 222 + 50% non-fat dry milk
894 = 100% non-germinated Vinton 81 (all soy yogurt- control)

949 = 100% non-fat dry milk (all dairy yogurt- control)

166

Mean values with different superscript are signiﬁcantly different (p < 0.05);

and within the same row (“3); n = 2.

 

The survival and viable numbers of probiotics in fermented dairy products are
different due to differences in strains and manufacturers (Champagne and others 2005).
In the present investigation, the survival of the probiotic i.e. L. acidophilus NCFM was
excellent during the 6-week cold storage. To effectively make some health claims on the
consumption of probiotics in fermented milk or soymilk, it has been suggested that the
cell numbers be a minimum of 6.0 log CFU/ml (Gomes and others 1998). All the
bacterial strains used in our study successfully attained a desired level of at least 7.0 log
CF U/ g of each strain in the yogurt samples. High proteolytic activity during storage being
suggested in this study could be due to the presence of hydrolysable substrate in the form
of milk and soymilk proteins.

Summarily, this investigation showed that whole soy-foritﬁed yogurt could be a
good medium for the delivery of probiotics. The data presented in this investigation
showed that germinated DF 222 soy-fortiﬁed yogurt was the best sample for the growth
and viability of the microorganisms used in this study. In general, all the cultures utilized

in yogurt manufacturing showed viability during prolonged cold storage at 4 °C.

167

CONCLUSIONS

. There was a signiﬁcant interaction (p_<_ .001) between non-fat dry milk/soy
powder blends, soybean variety and culture type, but NFDM/soy powder blend
was the most signiﬁcant factor (pg .001) that affected the activities and growth of

lactic acid bacteria.

. Cultures grown in most milk blends that contained whole soy powder gave the
lowest pH, highest %TA and growth (CFU/ml) values.

. Cultures grown in germinated whole soy powder blends produced more acid and
growth (pg .001) than its non-germinated counterparts suggesting that germinated
soybeans contained more bioavailable nutrients and other growth factors. Best
results were obtained when the cultures were grown in 50% non-fat dry milk +

50% germinated soy powder blend.

. All the cultures used in this study successfully attained a desired level, achieving
at least 107 CFU/ml of each strain in each blend.

. Overall data strongly support the view of several workers that non-fortiﬁed milk
does not enhance lactic acid bacteria and probiotics growth as much as fortiﬁed
milk.

. Fortiﬁcation of milk bases with whole soymilk or powder for fermented products
will enhance bioactive compounds and the viability of the cultures, hence

increases possible health beneﬁts to consumers.
. Sensory evaluation of soy-fortiﬁed yogurts showed no statistically signiﬁcant

difference between all-dairy yogurt and the 1:1 soy/NFDM blended yogurts for

ﬂavor and overall acceptance (p=0.0001). This suggests that there is no consumer

168

10.

11.

12.

13.

14.

preference for cow’s milk yogurt over whole soy-fortiﬁed cow’s milk yogurt at

this fortiﬁcation level.

One hundred percent soy yogurt had the lowest sensory scores than the other
yogurts in appearance and body texture although they were not signiﬁcantly
different. This could be attributed to the higher ﬁber content of the whole soy
powder utilized, which increased viscosity, and thus thickness.

Sensory data indicated that consumer acceptable soy-fortiﬁed yogurt could be
made using whole soymilk or powder blended with cow’s milk.

Nutrient composition analysis indicated that the protein contents of the entire soy-
fortiﬁed yogurt were comparable to the dairy yogurt. The fat contents were low in
all yogurt samples while all soy containing yogurts had dietary ﬁbers of up to
1.6% in 100% soy yogurt.

All the four isoﬂavone isomers measured in the soy powders were present but
genistin and daidzin, the B-glucosides were more abundant than genistein and
daidzein, the aglycones.

Soaking and germination increased the isoﬂavone contents and decreased the
stachyose contents of all soybean varieties, thus suggesting that processing

methods could inﬂuence the concentrations of these compounds.

The total isoﬂavone concentration was highest in non-germinated (soaked) DF
222 soy powder, but all the soy powders contain substantial amount of

isoﬂavones measured.

All the germinated soy powders irrespective of the variety had lower amounts of
stachyose. The lowest stachyose content was found in germinated E05276-T soy
powder (13.90 mg/ g) while the highest amount was found in non-germinated DF
222 soy powder (46.65 mg/g).

169

15.

l6.

17.

18.

19.

20.

Nutrient composition analysis showed that the protein contents of all soy powders
were between, 50.28% to 56.03% well above the average protein contents of

soybeans (40%).

Chemical analysis of yogurt samples showed that total isoﬂavone contents

increased at 6 weeks of storage (4 °C) compared to the 1St week. Daidzin and

genistin (B-glucosides) contents contributed most to the increment during storage.

The genistein and daidzein contents of the whole soy-fortiﬁed yo gurts remained
signiﬁcantly the same throughout the shelf life period (6 weeks at 4 °C). The
percentage of total isoﬂavones retained in the yogurt samples was high.

All the yogurt samples containing germinated soy powders had lower stachyose
contents than non-germinated soy-fortiﬁed yogurts (2.82 to 4.41 mg/ g of

stachyose).

The pH values of yogurt made with 100% non-germinated soy powder were
signiﬁcantly higher (p < 0.05) than the rest of the samples during storage.

The growth and viability of all the cultures were above 10 7 CFU/g of yogurt. A
concentration of at least 106 CF U/ml viable cultures in products is needed in order
to exert health beneﬁts to consumers. Soy-fortiﬁed yogurts had the highest viable

cell contents.

170

APPENDICES

171

APPENDIX 1

Questionnaire for experienced yogurt screeners

Product: Cow’s milk/soymilk blended Swiss style strawberry yogurt

You will be provided with 5 yogurt samples. Please carefully evaluate each sample
in the order it is presented and indicate the 3 most acceptable samples to you.

Acceptable = 1

Flavor

Body and Texture

Appearance and
Color

Overall Acceptance

817

817

817

817

172

Unacceptable = 0

626

626

626

626

978

978

978

978

149

149

149

149

481

481

481

481

APPENDIX 2

Advertisement

Do you like yogurt? Come taste...

.2

e.
E

LOW-FAT YOGURT

Dairy and Soy samples

Date: Thursday January 31“, 2008

Time: 11:00 am — 4:00 pm
Sensory Lab- Room 102 Trout (Food
Science) Building

 

Take 15-20 minutes to try some new

products and earn a MSU
ICE-CREAM coupon for helping out.

173

APPENDIX 3

MICHIGAN STATE Initial IRB

 

UNIVERSITY Application
Determination
September 17. 2007 *Exempt*

To: Zeynep USTUNOL
2105 8. Anthony Hall
MSU

Re: ' lees xor-ssr Category EXEMPT 1-6
ApproveiDete: September 14. 2007

Title: Yogurt fortification with predigestedlgerminated whole soybean powder for enhanced therapeutic
beneﬁts

ThelnstitutionalRevleoard hascompletedthelrrevioaofyowproiect lamplemedtoadviseyouthatyour
proiecthssbeendeemedesexemptmaccordancemmfederalreguadons.

The IRBhasfomdthetyourresearchprojectmeatsthecriteriaforexemptstatusandthecriteriaforthe
protection of human subjects in exempt research. Under our exempt policy the Principal investigator
assumes the responsibilities for the protection of human subjects in this project as outlined in the
assurance letter and exempt educational material. The IRB ofﬁce has received your signed assurance for
examptresearch. Acopyofthis signed agreementisappendedforyourinformation and records.

Renewals: Exempt protocols do mt need to be renewed. if the project is completed. please submit an
Application for Permanent Closure.

Revisions: Exemptprotocoisdongrequirerevislons. However.lichengesaremadetcaprotocolthatmayno
longer meet the exempt criteria. enewinltialapplicationwillberequired.

Problems: lfissues should arise during theconductofthe research. such as unanticipated problems. adverse
events.oranyproblemthatmayincressetherisktothehuman sublects andchangethecategoryofreview.
notiiythelRBcﬂicepromptly. Anycomplaintsirompanidpantsregardngthedskandbeneﬁtsoitheproiect
must be reported tothe IRB.

Follow-up: if your exempt project is not completed and closed after W. the "28 office will contact you
regardlngthestatusofthe project andtovarifythatnochanges haveoccurredthatmayaﬂectexemptstatus.

Please use the IRB number listed above on any forms submitted which relate to this project, or on any
correspondence with the RB ofﬁce.

Good luck in your research. lfwe can be of further assistance. please contact us at 517-355-2180 or via email
at W. Thank you for your cooperation.

Sincerely.
yeti-«€—

Peter Vasilenko. PhD.
BIRB Chair

174

APPENDIX 4

Consent form for panelists

SENSORY EVALUATION OF LOW-FAT YOGURT FORTIFIED WITH GERMINATED
WHOLE SOY POWDER
UNIVERSITY COMNIITTEE ON RESEARCH INVOLVING HUMAN SUBJECTS

TITLE OF THE RESEARCH
Yogurt fortiﬁcation with predigested/ germinated whole soybean powder for enhanced therapeutic beneﬁts.

INVITATION TO PARTICIPATE
You are invited to participate in this study, which compares the properties of yogurt made from cow’s milk
to those made from blends of cow’s milk and germinated soymilk.

PURPOSE OF THE STUDY
This study is being conducted in order to develop an acceptable yogurt made ﬁ'om blends of cow’s milk
and germinated soymilk.

BASIS FOR SUBJECT SELECTION

Subjects are selected based on their ability to detect differences in sensory attributes of yogurt made ﬁ'om
cow’ milk and from soymilk. Individuals with cold, sinus conditions or allergies to a speciﬁc ingredient
will not be asked to participate. The general adult population is used for testing. Participants must be at
least 18 years old.

POTENTIAL RISKS

The yogurt samples to be evaluated contain the following ingredients: milk, non-fat dry milk, soybean
powder, stabilizer, sweeteners (sucrose), strawberry puree, starter culture and red food color. All of these
ingredients are USDA and /or FDA approved for use in foods intended for human consumption and are
being used at USDA/FDA approved levels. Each product has being produced in a safe and wholesome
manner according to USDA and/or FDA regulations. These products samples pose no adverse health risk
upon ingestion, provided the subject has not been identiﬁed as being susceptible to an allergic reaction to
the previously listed product ingredients. If you believe there is a potential of an allergic reaction upon
ingesting the test products, or you believe that participating will violate religious or cultural beliefs, notify
the on-site sensory evaluation coordinator and/or principal investigator immediately. You will be released
from participating in the study.

POTENTIAL BENEFITS

There are no direct beneﬁts gained from participation in this study. However, your participation provides
valuable data for the development of products providing compounds with increased health beneﬁts.
Information obtained from this study will be published in appropriate scientiﬁc journals to expand our
current knowledge in enhancing the health values of yogurt.

EXPLANATION OF PROCEDURES

You will be asked to sit at a booth and taste a number of numerically coded yogurt samples. You will be
provided with water for rinsing your mouth between samples. The tasting exercise will take a maximum of
25 minutes of your time, depending upon your speed of tasting. You will use sensory evaluation ballot
forms to record responses concerning speciﬁc product attributes. Tasting will occur in the Sensory
Evaluation/Human Studies Laboratory located in Room 102 of the G. Malcolm Trout Good Science)
Building.

ASSURANCE OF CONFIDENTIALITY
Any information obtained in connection with this study that could be identiﬁed with you will be kept
conﬁdential by ensuring that all consent forms are securely stored and your privacy will be protected to the

175

maximum extent allowable by law. All data analyzed will be reported in an aggregate format that will not
permit associating subjects with speciﬁc responses or ﬁndings.

WITHDRAWAL FROM THIS STUDY

Participation in this study is voluntary. Your decision to refuse participation or discontinue participation
during this study will not affect your present or ﬁiture relationship with the principal investigator or
Michigan State University.

COMPENSATION FOR PARTICIPATION
After you have completed your sensory testing session and turned in your sensory ballot, you will be
offered a choice of treats (i.e., candy or ice cream coupon) for your time and effort.

OFFER TO ANSWER QUESTIONS

If you have any questions, please do not hesitate to contact the on-site sensory evaluation leader and /or the
principal investigator. You are voluntarily making a decision to participate in this study today. Your
signature certiﬁes that you have decided to participate after having read the information provided above
and that you had an adequate opportunity to discuss this study with the principal investigator and have had
all your questions answered to your satisfaction. You will be given a copy of this consent form for to keep
upon request.

SIGNATURE OF SUBJECT DATE

In my judgment the subject is voluntarily and knowingly giving informed consent and possesses the legal
capacity to give informed consent to participate in this research study.

SIGNATURE OF INVESTIGATOR DATE

Dr Zeynep Ustunol

2105 S. Anthony Hall

Food Science & Human Nutrition

Michigan State University, E. Lansing, MI 48824

Tel: 517-355-7713 ext 184, E-mail:_u_stunol@anr.nﬁr.edu

176

APPENDIX 5

Questionnaire
Product: Cow’s milk/soymilk blended strawberry ﬂavored low-fat yogurt
You will be provided with 6 yogurt samples followed by questions. Please evaluate
each sample in the order it is presented according to the scale provided.
1. Appearance/color

How do you like appearance and color of the sample

252 169 949 344 159 894

9- like extremely

8- like very much

7 - like moderately

6- like slightly

5- neither like/nor dislike
4- dislike slightly

3- dislike moderately

2- dislike very much

1- dislike extremely

2. Body and Texture
Take a scoop of the sample. Look at the sample and then taste it.

252 169 949 344 159 894

9- like extremely
8- like very much
7- like moderately
6- like slightly

177

5- neither like/nor dislike
4- dislike slightly

3- dislike moderately

2- dislike very much

1- dislike extremely

3. Flavor

How do you like the overall ﬂavor of the sample

252 169 949 344 159 894

9- like extremely

8- like very much

7- like moderately

6- like slightly

5- neither like/nor dislike
4- dislike slightly

3- dislike moderately

2- dislike very much

1- dislike extremely

4. Overall Acceptance

How well do you LIKE the sample overall?

252 169 949 344 159 894

9- like extremely
8- like very much
7- like moderately
6- like slightly

178

5- neither like/nor dislike
4- dislike slightly

3- dislike moderately

2- dislike very much

I- dislike extremely

Instruction:
You have completed the test. Thank you for your time. Don’t forget to ask for
your ICE CREAM COUPON before leaving!

179

REFERENCES

180

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